Tetramethylpyrazine (TMP), a flavor compound with biological activity, naturally exists in a variety of roasted, fried or fermented foods. Biosynthesis has been green and effective practice to produce TMP, and high TMP level was obtained in Bacillus subtilis BJ3-2 fermented dehulled adlay (BDA) in previous study. To elucidate the correlation between TMP and influencing factors in this study, the dynamics of TMP and influencing factors in BDA were investigated, and the correlation between TMP and influencing factors was analyzed by Pearson's correlation coefficients. Results showed that a weakly acidic environment was found in BDA and B. subtilis CICC 20637 fermented dehulled adlay (CDA), but a weakly alkaline environment was observed in B. subtilis BJ3-2 fermented soybean (BSB). The higher levels of α-acetolactate decarboxylase, reducing sugar, pyruvate, diacetyl, leucine, acetoin and TMP were detected in BDA, while higher levels of α-acetolactate synthase, free amino acids and ammonium were obtained in BSB. At the end of fermentation, the TMP content in BDA was 6.13 mg/g dry weight, which was 204- and 12-fold higher than that in BSB and CDA, respectively. TMP synthesis in BDA and CDA was remarkable correlation with pyruvate, diacetyl, leucine, ammonium and acetoin (p < 0.05). In summary, a weakly acidic environment, moderate ammonium content, high leucine and acetoin levels synergistically promoted TMP efficient synthesis.