Background Blood can only be given from generous donors. The main objective of this study was to assess the knowledge, attitude and practice (KAP) and associated factors of blood donation among health care workers in Wolaita Sodo University Teaching and Referral Hospital (WSUTRH), Wolaita Sodo, Ethiopia. Methods An institution-based cross-sectional study was conducted among 218 WSUTRH health care workers. Socio-demographic characteristics and data related to the levels of KAP of participants were collected using a self-administered questionnaire. Bivariate and multivariate logistic regression analyses were conducted using statistical package for social sciences version 20 to assess the factors associated with the practice of blood donation with p -value set at < 0.05 for statistical significance. Results Two hundred eighteen health care workers were involved in the study among which 129 (59.2%) were males and 89 (40.8%) were females. Among the study participants, 180(82.6%) had good knowledge but only 128(58.7%) were found to have a good attitude as 126(57.8%) reported that voluntary donor is the best source of blood donation. Regrettably, only 47(21.6%) of the respondents were found to practice blood donation in their lifetime. A majority (65.5%) of the participants did not donate blood as they have not been approached to do so. Knowledge and attitude levels of the participants were not found to be significantly associated with sociodemographic parameters study; but, only sex of the participants had shown statistically significant association with blood donation practice where males were more likely to donate blood than females (AOR = 2.59 (1.22–5.49)). Conclusions The overall level of knowledge was satisfactory and the level of attitude and practice was unexpectedly low. Female respondents were found to have lesser practice towards blood donation than males. Health care workers, blood banks and the hospital are demanded to design ways to update knowledge, and build its psychological benefits and make services more accessible. Electronic supplementary material The online version of this article (10.1186/s12878-019-0140-9) contains supplementary material, which is available to authorized users.