Characteristics and useful effects for human health of antioxidant carbon nanodots contained in microwave-assisted Opuntia humifusa extract (MA-OHE(CD)) were investigated in this work. MA-OHE(CD) was characterized using transmission electron microscopy, dynamic light scattering analysis, X-ray diffraction, UV–vis spectroscopy, X-ray photoelectron spectroscopy, and Fourier transform-infrared spectroscopy. Besides, total phenolic content and antioxidant activity of MA-OHE(CD) were measured. It was revealed that the MA-OHE(CD) increases aquaporin-3 expression in human epidermal keratinocytes cell with hydrophilic characteristics. Moreover, the bio-active compound extraction efficacy and antioxidant activity of microwave-assisted extraction were great when compared to maceration.