The invention discloses a construction method of Slco1b2 gene knockout rat and an application thereof. According to the invention, construction of Slco1b2 gene knockout rat is carried out by using a CRISPR/Cas9 system, which includes selection of Slco1b2 knockout targets, in-vitro synthesis and transcription of sgRNA and Cas9 mRNA, preparation of pseudopregnant mother rats, in-vitro microinjectionand transplantation of single cell embryos, rat breeding and screening, and obtaining of homozygote Slco1b2 gene knockout rat. Through verification by T7EI endonuclease, the gene knockout rat prepared by the method of the invention does not generate off-target phenomenon, and in the F2-generation knockout rat of about 4 weeks, total bilirubin, direct bilirubin and indirect bilirubin content are significantly higher than that of wild type rats. The invention also proposes an application of the construction method for hyperbilirubinemia research and drug development. The method provides an effective method for researching diseases such as increased bilirubin, and provides an effective experimental tool for drug research and development, and has broad application prospects.