Nickel oxide nanoparticles (NiO NPs) were synthesized via the facile sol-gel method. The synthesized NiO NPs were characterized using X-ray diffraction (XRD), Field-Emission Scanning Electron Microscopy (FESEM), Transmission Electron Microscopy (TEM), Raman and Fourier Transform Infrared (FT-IR) techniques. The effect of particle size was analyzed on structural alterations, electrochemical behaviors, and cytotoxic effect of synthesized NiO NPs. According to TEM results, the particle sizes of synthesized NiO NPs were 8.2, 15.4, and 21.7 nm at 300, 400, and 500°C, respectively. Electrochemical behaviors of synthesized NiO NPs were assessed through the cyclic voltammetry (CV) technique. The results showed that the magnitude of the current density was decreased in treated samples exposed to higher temperature values. The cytotoxic activity of synthesized nanoparticles was investigated against human liver cancer cell (Hep-G2), breast cancer cell (MCF-7), and colon cancer cell (HT-29) lines using the MTT assay. The results demonstrated that synthesized NiO NPs had higher cytotoxicity at 300˚C than at 400 and 500˚C, because of their small particle size. Thus, synthesized NiO NPs exhibit acceptable cytotoxic effects against Hep-G2, MCF-7, and HT-29 cancer cell lines so that they could be a good choice for cancer treatment.