This study provides a comprehensive characterization of V. alginolyticus isolated from diseased shrimp. A gram-negative, rod-shaped bacterium known as Vibrio alginolyticus was isolated. From the hepatopancreas of sick white leg shrimp also known as Litopenaeus vannamei provided by the University Malaysia Terengganu FPSM hatchery, bacteria were successfully isolated. Tests for isolates were conducted to ascertain their biochemical and physiological features. All of the biochemical and physiological characteristics are different, with the exception of salt tolerance testing, TCBS colony colour, and colony shape. Because certain isolates could haemolyse red blood cells, they were classified as virulent. The methods used to identify the bacterial strains included morphological and biochemical techniques, PCR, and sequencing of a conserved region of the 16S rRNA gene. The results showed that the isolates belonged to a single species. That grows in 1 to 10% NaCl, at 25 to 37°C, and on TCBS (thiosulfate-citrate-bile sucrose) agar. Non lactose fermenters and positive catalyse, the identification of V. alginolyticus was verified 99% the sequence identity of V. alginolyticus provided by the 16S rDNA sequence (GenBank accession number NR_PP980560.1). Via PCR analysis. The isolated strain's estimated LC50 dose was 1× 107 colony forming units (CFU/ml), and an inhibition zone was found around Penicillin (10 mm), Amoxicillin (5 mm), Chloramphenicol (15 mm), Tetracycline (1 mm), and Ampicillin (5 mm). Based on antibiotic susceptibility testing. The findings contribute to a better understanding of the virulence, resistance patterns, and ecological impact of V. alginolyticus, with implications for disease management in shrimp aquaculture.