Objective: To compare the applicability of fluorescent dye double-staining flow cytometry and firefly luciferase method for detecting CAR-T (Chimeric Antigen Receptor-T) cells cytotoxicity against target cells with different properties. Methods: For suspension target cells and adherent target cells, the fluorescent dye double-staining flow cytometry and the firefly luciferase method were used to detect CAR-T cell cytotoxicity, and The accuracy of the experimental results and the consistency of the trend were compared. Results: The fluorescent dye double-staining flow cytometry method is able to show significant difference of killing efficiency in the cytotoxicity experiment where the target cells are suspension (p<0.01), while the error is too large in the experiment where the target cells are adherent, resulting a non-significant difference (p>0.47); Firefly luciferase method is able to show significant difference of killing efficiency in both suspension and adherence target cell cytotoxicity experiments (p<0.01). Conclusion: The fluorescent dye double-staining flow cytometry method is more suitable for cytotoxicity experiments where the target cells are suspension, while the Firefly luciferase method is suitable for both suspension and adherent cells. Therefore, it is necessary to pay attention to method usage in specific applications.