Nonlinearity of electroosmotic flows (EOFs) is ubiquitous and plays a crucial role in ion transport, specimen mixing, electrochemistry reaction, and electric energy storage and utilization. When and how the transition from a linear regime to a nonlinear one occurs is essential for understanding, prohibiting, or utilizing nonlinear EOF. However, due to the lack of reliable experimental instruments with high spatial and temporal resolutions, the investigation of the onset of nonlinear EOF still remains in theory. Herein, we experimentally studied the velocity fluctuations of EOFs driven by an alternating current (AC) electric field via ultrasensitive fluorescent blinking tricks. The linear and nonlinear AC EOFs are successfully identified from both the time trace and energy spectra of velocity fluctuations. The transitional electric field (EA,C) is determined by both the convection velocity (U) and AC frequency (ff) as EA,C ∼ ff0.48-0.027U. We hope the current investigation could be essential in the development of both theory and applications of nonlinear EOFs.