Abstract: Provided are a preparation method for a CTL cell and an application thereof. The preparation method comprises the following steps of: inducing a CTL cell by using a tumor antigen PAP-GM-CSF sensitized DC cell; and knocking out a PD-1 gene of the CTL cell to obtain a PD-1 knock-out CTL cell. The CTL cell obtained by the preparation method can be used for preparing drugs for treatment of prostate cancer, especially for treating PAP-positive prostate cancer. The CTL cell does not cause CTL cell failure and anergy due to the tumor-expressed PD-L1 after being transfused into the body, thereby producing efficient specific cytotoxic effect on a tumor cell and improving the curative effect and reducing the side effect.