Background: Cervical cancer is the leading gynecological malignancy in Tanzania accounting 59.1 new cases per 100,000 women (age-standardized to the world population) and mortality of 42.7 deaths per 100,000 (age-standardized to the world population) in 2018. Despite this burden, the cervical cancer screening uptake is not effective. Currently, the proportion of cervical cancer screening is 11% in Tanzania among women but there is limited data of cervical cancer screening uptake among female health care workers especially doctors and nurses whom screening tendency among them might influence more screening uptake to the community since they are good messengers for disseminating health information to the community. The aim of this study was to assess the knowledge of cervical cancer, utilization, challenges, and factors associated with cervical cancer screening among female doctors and nurses. Methods: This was a hospital-based analytical cross-sectional study, conducted at Muhimbili National Hospital (MNH) from 1st August to 31st November 2021. The semi-structured self-administered questionnaires were used to collect data on knowledge of cervical cancer, screening uptake and challenges toward cervical cancer screening. These data were self-report. The factors associated with cervical cancer screening uptake were assessed using a multivariate robust poison regression model. A P< 0.05 was considered statistically significant. Results: A total number of 221 participants were involved in the study, 81.4% had good knowledge of cervical cancer and the proportion of self-reported utilization of cervical cancer screening was 29.9% (95% CI:23.9% – 36.4%). Lack of self-motivation toward screening (58.8%), tight work schedules (50.2%), and lack of perceived risk factors for cervical cancer (29.9%) were some of the challenges mentioned observed. The adjusted prevalence ratio (aPR) in the multivariate robust Poisson regression model revealed a lower level of education was associa