Vanillin (4-hydroxy-3-methoxybenzaldehyde), a food additive with rich milk flavor, is commonly used in the food, beverage and cosmetic industries. However, excessive consumption of vanillin may cause liver and kidney damage. Therefore, methods for detecting and controlling the level of vanillin in food, especially in infant powder, have important practical significance. In this study, we established a colorimetric assay for vanillin detection. The detection was performed under high-temperature and acidic conditions, which can induce the reaction of the aldehyde group of vanillin with the amino group of o-toluidine. The resulting product had a maximum absorption at 363 nm, which was quantified by a UV spectrophotometer. This assay had a limit of detection (LOD) of 1 pg mL-1 and a linear range between 1 μg mL-1 and 100 μg mL-1. The average recoveries at three spiked levels were in the range from 91.1% to 101.6% with a relative standard deviation (RSD) of 4.62% ~ 7.27%.