Objective Cerebral palsy (CP) is a prevalent neurodevelopmental disorder acquired during the perinatal period, with periventricular white matter injury (PWMI) serving as its primary pathological hallmark. PWMI is characterized by the loss of oligodendrocytes (OLs) and the disintegration of myelin sheaths, leading to impaired neural connectivity and motor dysfunction. Neural stem cells (NSCs) represent a promising regenerative source for replenishing lost OLs; however, conventional two-dimensional (2D) in vitro culture systems lack the three-dimensional (3D) physiological microenvironment. Microfluidic chip technology has emerged as a powerful tool to overcome this limitation by enabling precise spatial and temporal control over 3D microenvironmental conditions, including the establishment of stable concentration gradients of bioactive molecules. Catalpol, an iridoid glycoside derived from traditional medicinal plants, exhibits dual antioxidant and anti-apoptotic properties. Despite its therapeutic potential, the capacity of catalpol to drive NSC differentiation toward OLs under biomimetic 3D conditions, as well as the underlying molecular mechanisms, remains poorly understood. This study aims to develop a microfluidic-based 3D biomimetic platform to systematically investigate the concentration-dependent effects of catalpol on promoting NSCs-to-OLs differentiation and to elucidate the role of the caveolin-1 (Cav-1) signaling pathway in this process.Methods We developed a novel multiplexed microfluidic device featuring parallel microchannels with integrated gradient generators capable of establishing and maintaining precise linear concentration gradients (0-3 g/L catalpol) across 3D NSCs cultures. This platform facilitated the continuous perfusion culture of NSC-derived 3D spheroids, mimicking the dynamic in vivo microenvironment. Real-time cell viability was assessed using Calcein-AM/propidium iodide (PI) dual staining, with fluorescence imaging quantifying live/dead cell ratios. Oligodendrocyte differentiation was evaluated through quantitative reverse transcription polymerase chain reaction (qRT-PCR) for MBP and SOX10 gene expression, complemented by immunofluorescence staining to visualize corresponding protein changes. To dissect the molecular mechanism, the Cav-1-specific pharmacological inhibitor methyl-β-cyclodextrin (MCD) was employed to perturb the pathway, and its effects on differentiation markers were analyzed.Results Catalpol demonstrated excellent biocompatibility, with cell viability exceeding 96% across the entire tested concentration range (0-3 g/L), confirming its non-cytotoxic nature. At the optimal concentration of 0-3 g/L, catalpol significantly unregulated both MBP and SOX10 expression (P<0.05, P<0.01), indicating robust promotion of oligodendroglial differentiation. Intriguingly, Cav-1 mRNA expression was progressively downregulated during NSC differentiation into OLs. Further inhibition of Cav-1 with MCD further enhanced this effect, leading to a statistically significant increase in OL-specific gene expression (P<0.05, P<0.01), suggesting Cav-1 acts as a negative regulator of OLs differentiation.Conclusion This study established an integrated microfluidic gradient chip-3D NSC spheroid culture system, which combines the advantages of precise chemical gradient control with physiologically relevant 3D cell culture. The findings demonstrate that 3 g/L catalpol effectively suppresses Cav-1 signaling to drive NSC differentiation into functional OLs. This work not only provides novel insights into the Cav-1-dependent mechanisms of myelination but also delivers a scalable technological platform for future research on remyelination therapies, with potential applications in cerebral palsy and other white matter disorders. The platform’s modular design permits adaptation for screening other neurogenic compounds or investigating additional signaling pathways involved in OLs maturation.