Introduction Inappropriate self-medication results in wastage of resources, drug resistance and hence serious health risk. Knowledge acquisition and attitudinal change is required for avoiding self-medication practice. There is paucity of published articles regarding this topic. Therefore, this study aimed to study knowledge, Attitude and associated factors towards self-medication among health professionals at University of Gondar Comprehensive Specialized Hospital, northwest Ethiopia.Methods This cross-sectional study was conducted among health professionals of University of Gondar comprehensive specialized hospital using simple random sampling technique and self-administered questionnaire. We used Epi Info 7 and SPSS 20 for data entry and statistical analysis. Descriptive statistics were computed logistic regression had been performed to identify associated factors for knowledge and attitude separately. We executed crude and adjusted Odds ratio with 95% uncertainty interval. Variables with a p<0.05 were treated as significant. Results Four hundred and twelve health professionals participated in the study with 97.4% response rate. The mean age of study participants’ was 29.9 (±5.43, range; 20-60) years and 220 (53.4%) participants were males. From the participants 233(56.6%) (95% UI: 51.8-61.4%) had good knowledge about self-medication and 263 (63.8%) (95% UI: 59.2-68.4%) had good attitude towards self-medication. Knowledge of self-medication was associated with age of 32-60 years (AOR=2.19, UI: 1.14-4.22), Bachelor degree (AOR=6.49, UI: 1.86-22.69), Years after last graduation (AOR=2.49, UI: 1.41-4.41), working hours of 56-110 per week (AOR=2.35, UI: 1.18-4.67), and good attitude (AOR=2.95, UI: 1.77-4.91). Besides this, good attitude was associated with low workload out of workplace (AOR=2.78, UI: 1.03-7.50), ever taking antibiotics (AOR=2.45, UI: 1.31-4.50), and good knowledge (AOR= 2.64, UI: 1.65-4.20).Conclusion The study demonstrated that the knowledge and attitude of health professionals towards self-medication are relatively high. However still there is a need for health education on the implication and danger of self-medication. There is also need for government to increase awareness by creating training opportunity and incorporating in curricula.