Abstract: Some embodiments of the disclosure provide a method for detecting an anti-chimeric antigen receptor (CAR) antibody against humanized CAR T cells. In some examples, the method include following steps. S1. Transfecting a humanized CAR into Chinese hamster ovary (CHO) cells into using lentivirus to generate CHO cells expressing CAR as target cells. S2. Collecting samples to be tested. S3. Preparing different concentrations of standards using human proteins corresponding to a target of the CAR and establishing a standard curve. S4. Incubating the target cells with the different concentrations of standards and the samples to be tested, and capturing the anti-CAR antibody. S5. Incubating a phycoerythrin (PE)-conjugated anti-human IgG Fc antibody with CHO cells for labeling. S6. Calculating the anti-CAR antibody by detecting average fluorescence intensities of the standards, blank control, and the samples to be tested using flow cytometry.