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Introduction: Blood borne infections are one of the biggest obstacles in developing countries, destroying lives and livelihoods of millions of people due to reduced resources, limited education on infection control and fewer enforcement structures. Aims: This study was undertaken to evaluate the level of awareness, knowledge, and practice of nurses and laboratory technicians regarding blood borne infections. Methods: This prospective cross-sectional study was carried out by using pre-validated self-administered questionnaire and included a total of 570 respondents. Out of which 315 (55.26%) were nurses and 255 (44.74%) were laboratory technicians of tertiary care hospital of Gujarat. Result: The study found that the majority of the respondents are fairly aware about the general precautions, vaccination and post exposure prophylaxis (p value: >0.005) but have insufficient awareness regarding mode of transmissions and signs and symptoms of the blood borne infections (p Value: <0.05). There is not a significant variation between laboratory technicians and nurses in their understanding and awareness of blood-borne diseases. But nurses were found to be more knowledgeable and aware compared to the laboratory technicians (p value <0.05). Conclusion: Despite the fact that the majority of nurses and lab technicians had a fair understanding of blood borne diseases, this did not convert into effective adherence to universal protection measures and recommended practises. Our healthcare personnel must be immediately made aware of the risk of blood borne infections and inspired to follow safe working practises in order to avoid transmissions.