This study developed an innovative strategy for colouring meat products without nitrite addition, using nitric oxide synthase (NOS) fused with flavodoxin YkuN and flavodoxin reductase YumC derived from Bacillus subtilis. The results showed that the plasmids containing nos linked with ykuN and yumC genes by rigid linkers were constructed and chemically transformed into B. subtilis 168, and the enzyme fused with YkuN and YumC (NOS-YkuN-YumC) was successfully expressed and then purified. The activity of the fusion enzyme was approximately 12 times greater than that of NOS. In a model system, NOS-YkuN-YumC significantly increased the a*-value (redness) compared to those of the control and the sample treated with NOS (P <
0.05). UV-Vis spectral analysis indicated that metmyoglobin was converted to nitrosylmyoglobin (NO-Mb). In minced meat, the addition of NOS-YkuN-YumC significantly promoted the formation of NO-Mb and enhanced the a*-value (P <
0.05). The colour of the minced meat did not differ significantly between the NOS-YkuN-YumC group and the nitrite group (P >
0.05). This study provides a promising solution for enhancing colour formation in meat products without nitrite.