The source and synthesis ratio of VFAs were studied in this paper using isotope tracer technol. in view of the unclear source of volatile organic acids (VFAs) under anaerobic acid production and the lack of basis for directional synthesis. A complete anaerobic acid production system was constructed, in which bovine serum protein, vegetable oil and stable isotope glucose (99% abundance of C-13) were used as reaction substrates, and the source and proportion of VFAs were quant. analyzed by using the principle of carbon isotope tracing. The results showed that the carbohydrate (13C) would mainly promote the formation of even type short-chain fatty acids, with 77% and 86% contributions to acetic acid (HAc) and butyric acid (HBu), resp. However, the protein (12C) could be more conducive to the synthesis of odd type short-chain fatty acids, in which the contribution rates to propionic acid (HPr) and valeric acid (HVa) were 85% and 99%, resp. The concentration and ratio of odd and even short chain fatty acids in the hydrolyzate can be effectively adjusted by changing the ratio of carbohydrate and protein in the fermentation substrate, so as to provide a material basis for the later microbial synthesis of high-quality polyhydroxyfatty acid esters (PHA).