Breast cancer is the most often diagnosed and most common cause of cancer death worldwide, and it is reportedly very common among Ethiopian women. The aim of this study was to assess knowledge, attitude about breast cancer and breast cancer screening practice among female healthcare professionals from Bahir Dar, Ethiopia. Institution based cross-sectional study was employed from March 10, 2020 to April 2021 for a total sample of 259 female healthcare providers who are working in four selected healthcare units. Data were collected using a semi structured and pretested self-administered questionnaire, entered in EPI Info version 7 and analyzed using SPSS version 20 statistical software package. Adjusted odds ratio (AOR) with 95% confidence interval (95% CI) was used to assess statistical significance and P value ≤ 0.05 was used to determine level of statistical significance. The findings of this study revealed that only 123 (47.5%) of them had good knowledge about breast cancer and its screening and 136 (52.5%) had poor knowledge. Knowledge of breast cancer and breast cancer screening was found to be significantly associated with age, self- history of respondents, and marital status. Age ranging 36 years and above (AOR: 5.82; 95%CI: 1.49-22.6; P = 0.011), marital status with married (AOR: 3.63; 95%CI: 1.78-7.37; P < 0.001), and self-history of breast problem (AOR: 3.95; 95 %CI: 1.03-15.12; P = 0.045) were significant predictors of knowledge. The majority 190 (73.4%) of the respondents had a positive attitude towards breast cancer and breast cancer screening methods. Among those who heard about breast cancer 209 (80.7%) had practice of Breast Self –Examination (BSE), 98 (37.8%) said they had undergone clinical breast examination (CBE) and only 53(20.5%) had mammographic examination. It is recommended that the healthcare units should offer educative awareness programme to female healthcare professional regularly. Keywords: Knowledge, Attitude, Practice, Female healthcare professionals, Breast cancer, Breast cancer screening