Background. Hepatitis B virus (HBV) infection is the major infectious hazard for health care personnel. The global prevalence of HBV infection is highly heterogeneous, and the highest prevalence (6.2 and 6.1%) is among the World Health Organization Western Pacific and World Health Organization African regions, respectively. The pooled prevalence of HBV in Ethiopia among health workers was accounted for 5%. The prevalence rate of HBV in health care workers is about 2–10 times higher than the general population in the world. There for, the main aim of this study was to assess the knowledge, attitude and practice, and associated factors towards hepatitis B virus (HBV) infection among health care professionals at Tibebe Ghion Specialized Hospital, Bahir Dar, Northwest Ethiopia, 2021. Method. An institutional-based cross-sectional study design was at Tibebe Ghion Specialized Hospital, Bahir Dar, in 2021, and a systematic random sampling technique was used from different professionals, and the separate sample was taken independently from each. A pretested structured questionnaire was constructed and collects data then analyzed by using SPSS version 23. Result. A total of 422 health care workers having different professions have participated in this study. 243 (57.6%) of the study subjects were males. The average correctly answered knowledge, attitude, and practice questions were 65.6%, 40.3%, and 34.8, respectively. Multivariable logistic regression analysis showed that being nurse professionals (
A
O
R
=
0.17
(0.07, 0.38),
P
<
0.001
), midwives (
A
O
R
=
0.19
(0.07, 0.5),
P
=
0.001
), and work experience (
A
O
R
=
2.37
(1.38, 4.02),
P
=
0.002
) were associated with knowledge levels. Being degree holders (
A
O
R
=
2.49
(1.23, 5.02),
P
=
0.01
) and specialists (
A
O
R
=
9.78
(2.69, 35.5),
P
=
0.001
) were associated with attitude levels. Being medical laboratories (
A
O
R
=
17.42
(5.02, 60.5),
P
≤
0.001
) and pharmacy professionals (
A
O
R
=
11.2
(4.02, 31.42),
P
≤
0.001
) were associated with practice levels. Conclusion and Recommendation. Based on the current study, most of the health care professionals in this study area have poor knowledge, negative attitude, and malpractice towards HBV infection. Therefore, continual professional training programs on HBV infection include increased vaccination coverage rate and postexposure prophylaxis of heath care workers especially for highly exposed professionals.