The current study aimed to estimate the seroprevalence of SARS-CoV-2 IgG (S-protein) antibodies along with neutralizing assay (RBD-domain) among the whole blood donors without any prior Covid-19 history or symptoms visiting Blood Centre at a Tertiary care institution, South India amidst the ongoing pandemic. During September 2020 to March 2021, 1034 whole blood donors were enrolled into the study and were screened for anti-SARS-CoV-2 IgG antibodies using Chemiluminescence assay followed by neutralizing antibodies using surrogate neutralization ELISA. The study reported seroprevalence of 49.4%, (95% CI 46.3–52.5) among whole blood donors, with test sensitivity and specificity adjusted prevalence of 54.9% (95% CI 51.5–58.3). Seroprevalence was similar across age groups, gender, voluntary/replacement donations, area of residence, ABO and Rh groups without any statistical significance. However higher IgG antibody responses were found to be elicited in the 30–45 years age group when compared with 18–29 years age group (p value 0.046). This study also analysed the mean neutralizing capacity of SARS-CoV-2 antibodies among 97 blood donors which was 71.9 (SD: + 21.03, range 15.5 to 97.3). Donor samples with SARS-CoV-2 IgG S/Co > 9.5 had significantly higher neutralising capacity (> 68%) when compared with donor samples of S/Co < 9.5 (p value 0.000). Real-time seroprevalence studies will help to know the herd immunityamong the blood donors which will assist in knowing the Covid-19 transmission dynamics, distribution of immunity levels at a particular point in time, immunity gaps, development of novel therapeutics and prioritize the vaccination programmes to high risk individuals.