Objective Dust has steadily emerged as a frontier research in the field of forensic science because it is a material evidence with significant features and application potential that carries rich environmental DNA information. However, as a crucial foundational step in forensic applications, the collection and DNA extraction research of dust on object surfaces from the perspective of practical applications in forensic science are still in urgent need of development.Methods Dust was collected from object surfaces using a Copan Liquid Amies Elution Swab. DNA was extracted separately from the swab head, sediment, and supernatant within the sample collection tube to evaluate DNA content, thereby determining which components within the tube should be processed and lysed. Dust samples were collected according to five different sampling areas (25-400 cm2) and the DNA concentration was measured to determine the optimal sampling area. The extraction efficiency of three commercial DNA extraction kits for dust samples was compared. The size of the DNA fragments extracted from the dust was analyzed, as well as the presence of human DNA. Additionally, 16S rDNA amplicon sequencing was used to analyze the bacterial information in dust DNA from object surfaces. This process aimed to establish a quality control method for dust DNA extraction. Regarding the critical step of cell lysis in DNA extraction, the quantity of DNA extracted was compared and evaluated under different cell lysis methods and varying vortexing times. This was done to establish an appropriate cell lysis method for dust DNA extraction.Results The sediment and swab head in the dust sampling tube are the primary sources of DNA, and both should be included in subsequent extraction processes. The sampling area of dust is positively correlated with dust DNA concentration, and it is recommended that the sampling area be larger than 5×5 cm2. Using the DNeasy PowerSoil Pro kit can yield a higher amount of DNA. Additionally, there were no significant differences in the sizes of DNA fragments extracted by the three different DNA extraction kits. No human DNA was detected in the DNA extracted from the dust samples, while bacterial DNA was present in the dust from object surfaces. Furthermore, there were differences in microbial species composition between different sampling points. Additionally, using a biological sample homogenizer to grind and lyse for 4 min (2 min× 2 times) resulted in the highest concentration of dust DNA.Conclusion The extraction of dust DNA is influenced by the sampling area, extraction kits, and lysis methods. It is crucial to establish a comprehensive and suitable dust DNA extraction scheme. This not only lays the foundation for researching and extracting environmental DNA data from dust, but also provides a methodological reference for forensic case work involving environmental samples.