A method of detecting an RNA G-quadruplex is provided. The method includes bringing a sample to be detected to be in contact with cyanine dyes to obtain a mixed solution, subjecting the mixed solution to color observation or spectral analysis (including ultraviolet absorption spectrum analysis and fluorescence spectral analysis), and determining whether the RNA G-quadruplex exists in the sample to be detected based on color changes or spectral analysis results of the mixed solution. The method has characteristics of simple and convenient operation, high sensitivity and high accuracy.