Objective: To estimate the prevalence of co-occurrence of alcohol, tobacco, and illicit drug use among students at a public university in a municipality in Southern Brazil, and to evaluate the factors associated with this practice. Methods: Cross-sectional study conducted with undergraduate students between November 2017 and July 2018, to whom a digital self-administered questionnaire was applied. The relationship between the dependent variables co-occurrence of alcohol, tobacco, and illicit drug use and co-occurrence of alcohol and illicit drug use was analyzed using binary logistic regression with a 95% confidence interval. Results: Of the 1,824 university students, 92.0% consumed alcohol in their lifetime, 11.0% smoked, 43.9% used at least one illicit drug in their lifetime, and 23.3% used at least one illicit drug in the last 30 days. The rate of co-occurrence of alcohol, tobacco, and illicit drug use was 5.2%, and it was associated with belonging to the highest economic class (p = 0.0003), experimenting with drugs before the age of 18 (p = 0.0013), and attending an exact sciences or land/agricultural sciences program (p = 0.0386). The prevalence of co-occurrence of alcohol and illicit drug use was 13.6%, and it was associated with belonging to the highest economic class (p = 0.0000), male gender (p = 0.0323), depression (p = 0.0001), experimentation with alcohol before the age of 18 (p = 0.0000), attending a linguistics or arts and literature program (p = 0.0314) and living alone/with friends (p = 0.0000). Conclusion: The co-occurrence of alcohol and drug use was higher than the co-occurrence of use of the three substances, and factors such as experimenting with drugs before the age of 18 and being of higher economic status were associated with these patterns.