–Medical laboratories staff are exposed to professional bio-hazards on a regular basis if proper protective standards are not put in place, both their health and safety may be at danger. The initiative of this study is based on the articulation of the knowledge, attitude and practice regarding biological hazards among the laboratory staffs. Innumerable accidents happen due to lack of sufficient information about laboratory safety procedures, a callous attitude, and incorrect administration in the laboratory. This study was carried out among the staff of selected Hospitals and Diagnostic Centers at Jashore region to evaluate the KPA of laboratory bio-safety. In both approaches and manner, a descriptive cross-sectional survey was conducted among 192 laboratory staff from 52 Hospital and Diagnostic Centers. Data was gathered from a structured self-observed questionnaire and checklist, which were created and evaluated based on existing research and guidelines on universal work considerations. A total of 192 staffs who consented were included in the study. Where 150 (78.1%) male and 42(19.9%) female. 116 (64.4%) were 20-30 years age group, 49 (25.5%) were over 10 years of experience and 129(67.2%) married respondents. In the study 166 (86.5%) respondents work more than 8 hours in the lab and 59 (30.7%) were overweight. 66(34.4%) of staff have good knowledge on bio-safety, good attitude and practice 46 (24%) and 25(13%) on bio-safety respectively. Within the demographic information observed, only the working hour displayed significant (p=0.000) influence on the bio-safety knowledge. Age of the worker (p=0.000) and experience (p=0.001) had great influence on bio-safety attitude. Among the participants 104 (54.2%) received formal safety training and 155(80.7%) aware about universal work precaution. 145 (75.5%) never washed hands before putting gloves and 70(36.5%) always used PPE. Average (Mean ± SD) of laboratory staff knowledge, practice and attitude 71.72±10.40, 63.21±12.16 and 70.68±14.20 respectively. Overall, the data suggest that good knowledge and attitude to bio-safety practices between lab staff was indicated and their practices need to be more improved. Arrangement of training programs, proper monitoring and sticking to legislation must be implemented by the governance body to rise conversance of the laboratories staff about strict laboratory techniques and biohazards.