Background: Aflatoxin M1 (AFM1) in industrial ice cream was analyzed by immunoaffinity column (IAC) clean-up followed by high-performance liquid chromatography (HPLC) with a fluorescence detector (FLD) as a highly-sensitive method to confirm and quantify AFM1 in the ice cream samples. Methods: A total of 150 industrial ice cream samples were randomly collected from supermarkets in seven Iranian cities (Tehran, Tabriz, Karaj, Urmia, Sanandaj, Qazvin, and Khoramabad). Results: AFM1 was detected in more than 80.0% of samples. The average AFM1 concentration in ice cream was 29.79 ng/L. According to the results, in 12.62% of samples, AFM1 levels exceeded the maximum European Union (EU) limits for adults (50 ng/mL). This study recommends regular monitoring of AFM1 concentration in ice cream in Iran. Conclusion: Results of this study indicate the need to develop a database to assist law enforcement agencies, traders, and policymakers in local governments to minimize or prevent health-related risks caused by AFM1.