Accurate measurement of the gain and readout noise is very important for the miniature spectrometer performance evaluation and the spectrum data processing. Based on the noise principle of miniature spectrometers, the function between the output signal and the noise is deduced, and a method of measuring the gain and the readout noise of the miniature spectrometer is proposed. The measurement system is built according to the method. The gain of the self-developed miniature spectrometer is 2.02 e－/ADU, and the readout noise is 68.92 e－. Compared with the experimental results of the photon-transfer curve method and the measuring dark spectrum method, feasibility and effectiveness of the proposed method is further verified. The derivation process of this method is rigorous, and its operation process is simple. The method can be widely used in the measurement of gain and readout noise of the miniature spectrometer.