Cobalt nanoparticles (Co-NPs) have been extensively used in clinical practices and medical diagnosis. In this study, the potential toxicity effects of Co-NPs with special emphasis over the biochemical enzyme activities, such as aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) in serum, liver, and kidney of Wistar rats were investigated. This toxicity measurement of nanomaterials can support the toxicological data. The biochemical enzymatic variations are powerful tools for the assessment of toxicity. ASAT and ALAT enzymes have been widely used to predict tissue-specific toxicities associated with xenobiotic. The biochemical changes induced by Co-NPs have significance in their toxicological studies because the alterations in biochemical parameters before clinical symptoms indicate either their toxicant safety or detrimental effect. Herein, Co-NPs with particle size <50 nm significantly activated ASAT and ALAT enzymes in the serum, liver, and kidney of rats at concentration-dependent order.