Objective To develop a reliable method for the primary culture of airway smooth muscle cells (ASMCs) from mice by adherent tissue culture and to identify them by immunofluorescence microscopy. Methods Airway smooth muscle (ASM) tissue was isolated from BALB/c mice under dissecting microscope, and cut into 1 mm³ pieces. These tissue blocks were washed with PBS with 1% penicillin and streptomycin, and adhered on 6 cm culture dish with 5 mL culture media. The dishes were incubated at 37℃ in an incubator with 5% CO₂. The obtained primary ASMCs were identified by immunofluorescence microscopy, and the cell proliferation was measured by MTT assay.Results We observed that obvious fusiform cells grew out from tissue blocks within 3 to 5 days. After 5 to 6 days, “hill-valley” structure was observed. After cell passage and purification, the immunofluorescence microscopy showed that the purity of isolated ASMCs reached up to 99%. The growth curve of ASMCs was constructed by MTT assay. Conclusions Obtained ASMCs from this simple and economical method show a preferable proliferation ability, density and purity, and can satisfy the need for cell biology research.