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Pharmaceuticals in aquatic environment displayed adverse effects to fish. The effects are usually related to the internal levels of pharmaceuticals accumulated in specific fish tissues. In this study, we investigated the uptake, elimination, and toxicokinetics of six pharmaceuticals, e.g. naproxen (NAX), diclofenac (DCF), ibuprofen (IBU), carbamazepine (CBZ), fluoxetine (FLX), and sertraline (SER), in 11 fish tissues of Nile tilapia. The experiments were conducted in a flow-through system with an 8-day uptake/8-day elimination periods. The fish exposure groups involved the control, single FLX, and mixture of six pharmaceuticals at environmentally relevant concentration of 4 μg/L. FLX and SER showed the maximum concentrations of 145 and 201 ng/g wet weight, respectively, in fish spleen tissue, while NAX and IBU were not detected in any tissue. The mean concentrations for the pharmaceuticals in Nile tilapia tissues generally followed the order: bile> kidney, gut, stomach, liver> brain, gill, spleen> plasma, skin, muscle. The steady-state bioconcentration factors in various tissues generally range at 0.74–437.58 L/kg. The uptake and elimination toxicokinetics illustrated the rapid accumulation and depuration of pharmaceuticals in fish tissues. The results help to understand the internal bioconcentration, tissue distribution, and toxicokinetics of pharmaceuticals in multiple fish biological compartments.