Background: Aflatoxin is a toxic cancerous fungal secondary metabolite produced by Aspergillus species. Consumption of aflatoxin-contaminated cereals and their products can cause several health-related effects including acute and chronic aflatoxicosis. This study is aimed to evaluate the total aflatoxin content in Cheka and its major ingredients using the Immuno-Affinity Column Clean-up technique by sensitive HPLC with fluorescence detection. Methods:This study followed a completely randomized experimental design to evaluate the level of aflatoxin in traditionally brewed Cheka and its major ingredients (maize) by HPLC from November 2019 to June 2020. Traditionally brewed 6 Cheka beverage and 6 maize seed samples were collected randomly from Konso and transported to Bless Agri Food Laboratory in Addis Ababa for laboratory evaluation of aflatoxin in samples. The data were analyzed by SPSS version 25. Results: In the present study aflatoxin contamination was detected in all maize and Cheka samples. A mean total aflatoxin level of 6.78 ± 8.28 μg/kg and 10.76 ± 4.50 μg/kg were observed in maize and Cheka samples respectively. AFB1 predominantly contributed to the contamination of all samples. Although there was no significant difference while comparing between mean total aflatoxin of any other sample types, a significant difference was observed for the AFB1 mean of pitfall maize and their respective Cheka samples (P= 0.033). Conclusion: The mean level of AFB1 in pitfall maize differs from a respective Cheka sample. As most of the samples evaluated for aflatoxin contamination showed levels above healthy cut-off points of aflatoxin set by different authorities like the European Union, CODEX, and Ethiopian standards agency, there must be a protracted interventional approach to reduce aflatoxin contamination in maize and its products in the region.