Pollution of mycotoxigenic fungi and their toxins seriously affect the safety of grain, oil and food. The development and application of natural preservatives are important technologies and research areas in the field of food microbial control. Inhibitory effect of trans-2-methyl-2-pentenoic acid as a natural food flavor on the growth of Aspergillus flavus was determined in vitro by contact assay and in vivo in stored peanut seeds, with sorbic acid as a positive control. The results showed that trans-2-methyl-2-pentenoic acid exhibited a stronger inhibitory effect than sorbic acid. The MIC (Minimum Inhibition Concentration) values of trans-2-methyl-2-pentenoic acid and sorbic acid on A. flavus were 0.6 and 1.0 g/L, respectively. Moreover, trans-2-methyl-2-pentenoic acid had a 100% inhibitory effect on the control of A. flavus in stored peanut seeds at 10.0 g/L, while a higher concentration of 20.0 g/L was needed for sorbic acid to achieve the same control effect. The results indicate that trans-2-methyl-2-pentenoic acid as a natural food flavor shows significant antifungal effect, and has high potential for the development of natural food preservatives.