Introduction: Following emergency approval of COVID-19 vaccines, several studies have investigated COVID-19 vaccine acceptance and hesitancy especially among healthcare workers (HCWs). Nevertheless, the acceptability of the COVID-19 vaccine by HCWs in Sudan remains unclear. This study aims to investigate the acceptability of the COVID-19 vaccine and it’s determinants among the HCWs. Methods: A web-based cross sectional study design was used to study COVID-19 vaccine hesitancy and its associated determinants. The semi-structured questionnaire was distributed electronically, data collection took place from March-April 2021. Results: A total of 576 HCWs have responded to the survey with mean age of 35 years. The majority were females (53.3%), Medical Doctors (55.4%), and located in the capital state; Khartoum (76.0%). The absolute refusal of COVID-19 vaccine was expressed by 16% of the respondents while 57% were willing to get vaccinated. Males were twice to four times more likely to accept the vaccine. Lower acceptability of COVID-19 vaccine was significantly associated with the nursing profession (OR= 0.35, 95% CI:0.15-0.82, P<0.00), increased perceived harm from the vaccine (OR= 0.11, 95% CI: 0.05-0.23, P <0.00), lack of confidence in the source of vaccine (OR= 0.16, 95% CI:0.08-0.31, P=0.00), organizations and government supervising COVID-19 vaccination process ( OR=0.31, 95% CI:0.17-0.58 P=0.00). Conclusion: This study highlights a moderate level of COVID-19 vaccine acceptability by the HCWs in Sudan. Effective communication of correct, regular, up-to-date evidence on the safety and effectiveness of vaccines is crucial to building trust in vaccines. Special consideration should be in place to address vaccine hesitancy among female HCWs and the nursing profession.