The most important mode of transmission causing outbreaks of Chagas disease in the Amazon region is the oral route due to the ingestion of contaminated food. Herein, prevention methods for foodborne diseases caused by Trypanosoma cruzi, namely, sanitization, thermal treatment were investigated and the use of reverse transcription PCR (RT-PCR) amplification for the mRNA-based detection of viable T. cruzi in açai, was developed. Three T. cruzi strains (T. cruzi I, T. cruzi III and Y) were used in the present study. The Amazonian strains T. cruzi I (425) and T. cruzi III (370) showed higher resistance to sodium hypochlorite treatment and heat treatment than the reference strain Y. The blanching of fruits (70 ± 1 °C for 10 s) and pasteurization of juice (82.5 °C for 1 min) efficiently eliminated T. cruzi in food matrices. Additionally, a method that uses RT-PCR amplification of mRNA was developed for the detection of viable T. cruzi in açai, which could play a role in examining food samples, ensuring consumer health, and reducing this foodborne disease.