Objective To construct a novel electrochemical aptasensor for L-lactate detection.Methods Based on gold-palladium-doped nitrogen-rich multi-walled carbon nanotube nanocomposite (Au/Pd-N-MWCNTs) modified glassy carbon electrodes, a triple-helix molecular switch (THMS) triggers the cyclic shearing effect of Pb²⁺-assisted deoxyribozyme (DNAzyme) with RNA-cleaving activity on the signal probes that are loaded on the electrode surface, used for ultrasensitive electrochemical detection of L-lactate. Differential pulse voltammetry (DPV) records the current signal changes.Results The signal probes concentration of 4 μmol/L, the Pb²⁺ concentration of 4 μmol/L, and the DNAzyme shear incubation time of 60 min are the optimal test conditions for the sensor. Under optimal experimental conditions, the L-lactate sensor’s linear range is 1-20 mmol/L, and the detection limit is 0.51 mmol/L. In addition, the aptasensor has excellent stability (RSD=1.04%), reproducibility (RSD=2.80%) and selectivity. When detecting L-lactate in human serum samples, the recoveries were 105.60%- 110.80%, and the RSDs were 2.35%-4.56%, which has high consistency with the traditional method.Conclusion The aptasensor can realize ultrasensitive L-lactate detection and has broad application prospects in biomedical diagnosis, the food industry, and environmental monitoring.