Abstract: Vanillin is classified into natural vanillin (i.e., the vanillin extracted from vanilla beans), chemically synthesized vanillin, and biologically synthesized vanillin. The prices of vanillin derived from different sources vary greatly, so identifying the source of vanillin can help to combat persistent fraudulent activities. Owing to the differences in isotopic compositions of vanillin from different sources, isotope technology is effective for distinguishing natural vanillin from biologically or chemically synthesized vanillin. In this study, the δ¹³C、δ²H、δ¹⁸O, and ¹⁴C activity of vanillin from different precursor materials and synthesis pathways were analyzed, and then one-way analysis of variance was conducted to identify the isotopic characteristics of vanillin from different precursors and synthesis pathways, verifying the feasibility of tracing the sources of vanillin using isotope technology and providing basic data for the vanillin isotopic database. Additionally, for the first time, the influence of using water with different isotopic compositions during the preparation processes on the vanillin’s isotopic compositions was investigated, thereby further improving the vanillin isotope tracing methodology. The results showed that the combination of δ¹³C and δ²H can clearly distinguish vanillin derived from different precursor materials including natural eugenol, ferulic acid, 4-methyl-guaiacol and so on. The combination of δ²H and δ¹⁸O can also distinguish vanillin derived from different precursor materials, but the differentiation effect is inferior, mainly because the discriminatory effect of δ¹⁸O is less clear. The activity of radioactive isotope ¹⁴C can identify whether the precursor material of vanillin is from natural biological sources or fossil fuels, while it is hard to distinguish the specific biological origin of vanillin, even if they come from different plant precursors. However, these four indicators can not distinguish vanillin from different synthetic routes (e.g., chemical or biological synthesis). Meanwhile, the water used in the production process has little effect on the δ²H and δ¹⁸O of vanillin, but the use of crystallization solvents and enzymes during the processes has a significant impact on the δ²H and δ¹⁸O of vanillin. These results indicated that the precursor materials, the exchange reactions during the biochemical synthesis or purification processes, and the degree of isotopic fractionation may all have influence on the isotope distribution in the final product of vanillin. As a result, the stable and radioactive isotope techniques are effective tools for identifying the source of vanillin, which can provide strong technical support for combating vanillin fraud.