This study proposes the nanobubble isotope separation method for the first time. The separation of light elements such as hydrogen, oxygen, carbon, and lithium is realized via experiments, and the separation coefficient is measured, which verifies the scientificity and effectiveness of the method. The study revealed that the isotope separation process of nanobubbles not only occurs when the rapid collapse adiabatic self-shrinkage forms nanobubbles, which causes the dissociation of surface molecules possibly owing to high temperature or nano-surface effects, such that the surface of the bubbles is negatively charged and adsorbs the surrounding medium, but also occurs in the subsequent isotope (ion) chemical exchange process between nanobubbles and specific solutions to form a separation system, which has a dual separation effect. Because the formation of nanobubbles is a rapid process, and the ion exchange between bubbles and solution is an isotopic resonance exchange chemical reaction, the process also quickly reaches equilibrium. The bubbles and solution determine that nanobubble isotope separation is a separation method with a short equilibrium time, overcoming the shortcomings of the usual chemical method balance time. Based on the prototype stand-alone machine for nanobubble separation, nanobubble isotope separation cascades are also designed to increase the separation effect to obtain isotopes of various abundances, thereby illustrating the possibility of industrial production.