To explore the feasibility of the ATP bioluminescence tumor chemosensitivity assay (ATP-TCA) applied in cervical squamous cell carcinoma chemosensitivity for testing and screening the sensitive drugs, and investigate the relation between chemotherapy-sensitive spectrum of cervical squamous cell carcinoma and clinicopathological parameters. ATP-TCA was used to detect the sensitivity of 34 specimens of fresh cervical squamous cell carcinoma to 11 cytotoxic drugs (16 grapes). Thirty-two assays were completed successfully, with an evaluability rate of 94.12% (32/34). There was a marked heterogeneity of chemosensitivity in cervical squamous cell carcinoma, and no significant association was found between the sensitivity and clinicopathologic feature such as clinical stage, organization type and cell differentiation. The in vitro sensitive rate of PTX+CBP, MMC+5-FU, GEM+DDP, 5-FU+DDP were 71.88% (23/32), 62.50% (20/32), 56.25% (18/32), 53.12% (17/32). ATP-TCA can be used to individualize chemotherapy by selecting agents for particular patients in cervical squamous cell carcinoma. The sensitive rates of PTX+CBP, MMC+5-FU, GEM+DDP and 5-FU+DDP are higher than other cytotoxic drugs.