The invention relates to an application of DHFR in bladder cancer detection, and belongs to the technical field of molecular biology gene detection. The application includes the following steps: 1, respectively extracting DNA from the bladder tissue and peripheral blood of a sample to obtain DNA templates; 2, respectively carrying out real-time quantitative PCR amplification on the DNA extracted from the bladder tissue and peripheral blood of the sample with a primer pair P as a primer; and 3, calculating the log2 ratio of the sample according to delta delta Ct, and comparing the log2 ratio with copy number variation obtained by using whole genome sequencing data, and determining as a bladder cancer sample when the copy number variation is more than 2.5 or is less than 1.5 or when a G fraction obtained through an GISTIC algorithm is more than 0.08 or is less than 0.08. In the invention, sequence amplified DHFR can be used as a marker for the bladder cancer detection; and DHFR gene can be used as a gene target treatment kit of the bladder cancer.