Transglutaminase (EC 2.3.2.13, TGase) recorded the highest activity (0.101 U/ml) in bacterial isolate NRC215. 16S rRNA sequencing revealed that NRC215 was identified as Bacillus cereus NRC215 under accession number MT229271 in the NCBI database. UV irradiation was employed to improve TGase production. Five rifampin (RIF) resistant mutants were only isolated from UV-treated Bacillus cereus NRC215 for three minutes. The best mutant, BCrif5, exhibiting induced rifampin resistance, gave TGase with higher activity (0.148 U/ml). The ISSR PCR technique was employed to detect these new rearrangements resulting from UV mutagenesis between the wild-type strain and its mutants. Moreover, TGase has been purified by three-step procedures resulting in a recovery of 28 and 34.63% for wild and BCrif5 strains, respectively. The optimal purified TGase activity was exhibited at pH 7 for wild strain while the mutant BCrif5 at pH 5.0 and 40 °C for both wild and BCrif5 strains. Bacillus cereus NRC215 TGase was activated by Ba+2 (102.50 and 107.06%), while it was inhibited by Cu+2 (30% and 22.35%) for wild and BCrif5 strains, respectively. It could be concluded that Bacillus cereus NRC215 is a promising strain for TGase production, which is beneficial as a food additive.