 In this study, the structure and stability of acid phosphatase in its interaction with Co3O4 nanoparticles are evaluated through absorbance, enzyme activity assay, fluorescence, thermal stability, and circular dichroism spectroscopy. It had been found that with the enhancement of Co3O4 nanoparticle concentration, the intensity of the enzyme’s ultraviolet spectrum was increasing. In fact, under these conditions (pH 4.8, T = 310 K), the activity of acid phosphatase was raised too. Also, it was revealed that by enhancing the concentration of the nanoparticles, the enzyme thermal stability increased from 342.0 to 346.0 K. Also, far-UV CD investigations illustrated that the Co3O4 nanoparticles could alter the secondary structure of acid phosphatase through an increase in the value of the α-helix structure (from 10.8 to 13.9%) and a decrease in the β-sheet (from 30.2 to 28.0%). By raising the temperature from 298 to 308 K, the Stern–Volmer constant decreased from 5.72 × 104 to 4.39 × 104 M−1. Also, it was found which Co3O4 nanoparticles quenched the intrinsic fluorescence of acid phosphatase by the static quenching mechanism. Therefore, the thermodynamic parameters showed that the binding process was spontaneous because the value of ∆G was negative. Also, van der Waals forces and hydrogen bonding interactions had the main effects on the interaction of Co3O4 nanoparticles with acid phosphatase because the values of ∆H and ∆H were negative. So, Co3O4 nanoparticles increased the stability and activity of acid phosphatase.