In order to study the optimal enzymatic hydrolysis process for preparing thermal reaction bovine bone flavoring, bovine bone extract (BBE) was used as raw material, and compound enzyme was selected. The effects of enzymatic hydrolysis temperature, enzymatic hydrolysis time, compound enzyme addition amount and compound enzyme addition ratio on the degree of hydrolysis of enzymatic hydrolysate and the sensory score of thermal reaction bovine bone flavoring prepared by Maillard reaction of enzymatic hydrolysate with sugar and amino acid were investigated by single factor and response surface test. The mass ratio of free amino acid was determined by high performance liquid chromatography (HPLC), and the mass ratio of key odor compounds of thermal reaction bovine bone flavoring prepared before and after enzymatic hydrolysis of BBE was analyzed by comprehensive two-dimensional gas chromatography-olfactometry-mass spectrometry (GC×GC-O-MS). The results showed that the better enzymatic hydrolysis process of BBE was enzymatic hydrolysis temperature of 50°C, enzymatic hydrolysis time of 4 h, enzyme addition amount of 1250 U/g, and the ratio of compound flavor protease to compound protease was 1: 1. Under this condition, the sensory score was 8.24. Compared with BBE, the mass ratio of 15 free amino acids in the enzymatic hydrolysate increased significantly. The odor profile and taste profile of BBE and enzymatic hydrolysate were measured by electronic nose and electronic tongue. It was found that the nitrogen oxide odor characteristics of enzymatic hydrolysate were enhanced, and the umami, richness and salty taste characteristics of enzymatic hydrolysate were more obvious. The key odor compounds of thermal reaction bovine bone flavoring prepared by BBE and enzymatic hydrolysate were analyzed by solid phase microextraction (SPME) combined with GC×GC-O-MS. A total of 164 volatile odor compounds were identified, including 27 aldehydes, 30 ketones, 12 alcohols, 16 acids, 7 sulfur-containing compounds, 10 esters, 18 pyrazines, 10 furans, 6 thiazoles, 4 thiophenes and 24 other compounds. Through gas chromatography-olfactometry (GC-O) analysis, 74 odor-active compounds were detected. There were 22 key odor compounds with odor activity value (OAV) greater than 1. The most important key odor compounds in the thermal reaction bovine bone flavoring prepared by BBE and enzymatic hydrolysate were bis(2-methyl-3-furanyl) disulfide (OAV was 13078 and 8052, respectively). Compared with the thermal reaction bovine bone flavoring prepared by BBE, 5 new key odor compounds (E,E)-2,4-decadienal, 2-methyl-3-furanthiol, methyl furfuryl disulfide, S-methyl thiohexanoate and β-caryophyllene appeared in the thermal reaction bovine bone flavoring prepared by enzymatic hydrolysate. The purpose of this study was to provide a theoretical basis for the enzymatic hydrolysis process in the industrial production of thermal reaction bovine bone flavoring.