Streptococcus iniae is a gram-positive, sphere-shape and pathogenic bacterium that usually causes infections in fishes as well as human. Glutamate racemase is a key enzyme that is involve in synthesis of peptidoglycan layer of bacteria cell wall. Biochemical properties and nature of this enzyme are highly specific and varies among pathogenic species. The unique features structural, molecular and biochemical properties associated with MurI gene as well as functional properties of glutamate racemase in the pathogenicity of S.iniae remain unclear. The purpose of this review paper focusing on the methods that used for the evaluation of biochemical properties of glutamate racemase for functioning in bacterial cell wall focusing on MurI Gene in S.iniae as well knockout of MurI gene in S.iniae also the discussing the methods used for the determination for the biochemical properties associated with pathogenicity at molecular level. Specific primers are used for the gene knockout plasmid construction region for MurI gene of bacterial strain such as S.iniae. These are further amplified by the PCR. DNA sequencing is use to clone the DNA fragments. The LB medium is the medium especially use for the S.iniae wild type (WT) and ΔMurI mutant cultures. Bioscreen machine is use to measure the optical density. The special type of the microscope such as fluorescence microscope is uses for the bacterial viability. TSYE agar plates are used for the measurement of the live bacteria in the form of percentage by plating on. SDS-PAGE is uses to check the purity of expressed proteins determined by the SDS-PAGE. This review helpful for drug discovery and development of new antibiotics by utilizing the MurI gene as a target against S.iniae.