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Malachite green (MG) is prohibited in fish farming in Vietnam due to its toxicological and carcinogenic properties. However, it continues to be illegally used in some areas. This study investigated the accumulation and depuration of MG in three organs—gills, liver, and muscle—of Oreochromis mossambicus (Tilapia), which represent the major toxicant entry routes: respiratory, digestive, and dermal, respectively. Under controlled laboratory conditions, the concentrations of MG and its primary metabolite, the reduced and colorless leucomalachite green (LMG), were analyzed using liquid chromatography–mass spectrometry. The results showed that MG primarily accumulated in the gills, with the lowest accumulation observed in muscle tissue. These findings suggest that tilapia have the ability to eliminate toxicants from their bodies. The fish demonstrated gradual adaptation to environments containing MG, with enhanced detoxification via excretory pathways over time. Notably, fish exposed to low MG concentrations were better able to adapt and eliminate the toxicant compared to those exposed to high concentrations continuously, indicating that exposure level plays a crucial role in the efficiency of detoxification.