The present application discloses a method, kit and application for screening target regions detected by methylation PCR. The method of the present application includes (1) obtaining the methylation chip of the tumor to be analyzed and the corresponding transcriptome sequencing data from the database; (2) counting the methylation degree values of the normal group and the cancer group, and screening for significant differences among different groups (3) Combined transcriptome sequencing expression profile analysis, statistical correlation coefficient, and screening of negatively correlated sites; (4) Correlation between the methylation candidate sites obtained in step (3) and the disclosed Literature association, screen to obtain sites with many literature support reports, large methylation differences between groups, and negatively correlated expression levels; use regression algorithm to obtain the best sensitivity and specificity set of sites, that is, the target area. The method of the present application comprehensively analyzes databases, transcriptome sequencing and literature, and combines multiple data filtering and regression algorithms to obtain sensitive and specific methylation PCR detection target regions.