Introduction: Healthcare Workers (HCWs) are at a high-risk of acquiring Hepatitis B Virus (HBV) infection. However, this risk can be prevented through Hepatitis B vaccination. In some institutes, HCWs have a lower percentage of HBV vaccination, leading to a higher risk of HBV transmission. Therefore, the coverage of vaccination is an important point, along with the evaluation of protective immune status. Aim: To assess the vaccination coverage and evaluate the immune response post-vaccination through Anti-Hepatitis B surface Antibodies (Anti-HBs) titre. Materials and Methods: This cross-sectional study was conducted at Government Medical College, Pali, Rajasthan, India, over a period of one year from April 2021 to March 2022, following approval from the Institutional Ethics Committee (IEC). A total of 455 HCWs below 60 years of age were included in the study, and their demographic details such as age, gender, occupation, needle stick injury, blood exposure to mucous membranes and breached skin, hepatitis B vaccination status, and time duration since vaccination were noted. Additionally, their Anti-HBs titer was examined. The participants were initially classified into three groups: completely vaccinated, partially vaccinated, and non vaccinated. Among those who were vaccinated, they were further divided into two groups: vaccination ≤5 years (Group A) and >5 years (Group B). Furthermore, those who received a booster dose were divided into Group I (≤1 year) and Group II (>1 year). Blood samples were collected to assess the anti-HBs levels quantitatively in the sera using Enzyme Linked Immuno Sorbent Assay (ELISA). The data was entered into Microsoft Excel and later imported into Statistical Package for Social Sciences (SPSS) version 22.0 for statistical analysis. Results: Participants had a mean age of 29.65±9.603 years. Among them, 43 (30.1%) were doctors, and 37 (25.9%) were medical students who were found to be completely vaccinated. On the other hand, among other HCWs, 15 (10.5%) were nurses, 16 (11.2%) were nursing students, and 32 (22.4%) were lab technicians who were vaccinated. None of the support staff were vaccinated (p-value=0.0001). A total of 143 participants were completely vaccinated, and 97.9% (n=140) had protective immunity to Hepatitis B. The anti-Hbs titre was 567.32±434.494 in group A and 265.74± 211.80 in group B (p-value=0.0001). Similarly, it was 688.34±424.617 in group I and 221.14±141.221 in group II (p-value <0.0001).The anti-Hbs titre did not significantly increase among the partially vaccinated participants (n=162). It was found to be 25.47±27.595 in group A and 14.60±19.939 in group B (p-value=0.004). There was no significant difference in the results between males and females (p-value=0.961). Conclusion: The coverage of complete vaccination among HCWs was significantly low, which is crucial for obtaining a protective Anti-Hbs titre. Incomplete vaccination does not result in a sufficient level of anti-Hbs titre, and there may be a significant decline in the immune response over time (p-value<0.05). Therefore, it is essential to estimate the titre after 1-2 months of complete vaccination to ensure that individuals are fully protected against Hepatitis B.