The invention discloses a kit for measuring content of total bile acid of blood serum and a use method of the kit, relates to the field of biochemical detection and aims to provide a high-stability, high-accuracy and non-interfering kit for measuring the content of total bile acid of blood serum and a use method of the kit. The kit comprises a reagent 1 and a reagent 2, wherein the reagent 1 comprises a GOOD'S buffer solution, thionicotinamide adenine dinucleotide, polyoxyethylene ether of nonylphenol, polyoxyethylene lauryl ether, ADP.2Na, EDTA (ethylene diamine tetraacetic acid), bovine serum albumin, mannitol and a preservative (proclin series); the reagent 2 comprises a GOOD'S buffer solution, 3 alpha-hydroxysteroid dehydrogenase, reduced coenzyme and the like. Due to addition of ethylene glycol, sucrose and multiple compound stabilizers, the stability of the reagents is improved; due to addition of EDTA, the interference effect of metal ions can be reduced and the test result accuracy can be improved.