The invention relates to a bioluminescence detection kit for hCE1 (human carboxylesterase 1) as well as a use method and an application of the bioluminescence detection kit. The kit comprises a solution A, a solution B, a solution C and a quality control standard, wherein the solution A is 100 mM of a phosphate buffer solution with pH of 5.5-10; the solution B is 0.1-10 mM of a solution containing a specific bioluminescent probe substrate of hCE1; the solution C is a fluorescein detection reagent; the quality control standard is a hCE1 phosphate solution with concentration of 5 mg/ml and pH of 6.5. The probe substrate is (S)-2-(2-(6-dimethylamino)-benzothiazole)-4,5-thiazoline-4-methyl formate and can be catalyzed specifically with hCE1 to be subjected to an ester bond hydrolysis reaction, and activity of hCE1 in various biological samples is determined by quantitatively detecting the generation amount of ester bond hydrolysis products of the probe substrate in unit time. The kit can be used for detecting whether hCE1 is present in different biological samples and quantitatively determining the activity of hCE1 and can be used for rapidly screening an inhibitor/inducer of hCE1. The bioluminescence detection kit has the advantages of being simple to operate, low in unit detection cost, sensitive and rapid, and high-throughput detection can be realized.