A serological test for screening of TiLV in Oreochromis niloticus would be useful for the epidemiological investigations. Using polyclonal antisera against TiLV (TiLV-Ab), an indirect enzyme-linked immune sorbent assay (iELISA) was developed for the detection of TiLV antigen in fish tissue and mucus. After a cutoff value was established and antigen and antibody concentrations were optimized, the iELISA's sensitivity and specificity were assessed. We found the ideal dilutions of TiLV-Ab as 1: 4000 and secondary antibody as 1:65,000. High analytical sensitivity and moderate specificity were displayed by the developed iELISA. The Positive and Negative Likelihood Ratio (LR+, LR-) were 1.75 and 0.29, respectively. The estimated Positive and Negative Predictive Values (PPV and NPV) of the test were 76.19% and 65.62%, respectively. The accuracy of the developed iELISA was estimated as 73.28%. An immunological survey was performed using the developed iELISA with samples from the field and 155/195 fishes tested positive, indicating a 79.48% TiLV antigen positives. Among the pooled organs and mucus tested, the highest positive rate of 92.3% (36/39) is observed in mucus compared to other tissues, and least positive rate is found in liver of 46% (18/39). The newly designed iELISA proved sensitive and may be helpful for extensive examinations of TiLV infections and monitoring disease status even from apparently healthy samples using a non-invasive technology by collecting mucus as sample for iELISA. •Using TiLV-Ab, a sensitive iELISA for the direct detection of TiLV antigen in fish tissue and mucus samples was developed.•The estimated PPV and NPV of the test were 76.19% and 65.62%, respectively, and the accuracy was estimated as 73.28%..•The highest TiLV positive rate is observed in the mucus samples compared to tissues (kidney, liver, and brain)..•Extensive examination of infection can be done even from apparently healthy fishes using non-invasive collection of mucus.