Effective cultivation of the microalgae is the key issue for microalgal bio-energy utilization. In nutrient rich culture conditions, the microalge have a fast growth rate, but they are more susceptible to environmental pollution and influence. So to monitor the the growth process of microalgae is significant during cultivating. Hyperspectral imaging has the advantages of both spectra and image analysis. The spectra contain abundant material quality signal and the image contains abundant spatial information of the material about the chemical distribution. It can achieve the rapid information acquisition and access a large amount of data. In this paper, the authors collected the hyperspectral images of forty-five samples of Chlorella sp., Isochrysis galbana, and Spirulina sp., respectively. The average spectra of the region of interest (ROI) were extracted. After applying successive projection algorithm (SPA), the authors established the multiple linear regression (MLR) model with the spectra and corresponding biomass of 30 samples, 15 samples were used as the prediction set. For Chlorella sp., Isochrysis galbana, and Spirulina sp., the correlation coefficient of prediction (rpre) are 0.950, 0.969 and 0.961, the root mean square error of prediction (RMSEP) for 0.010 2, 0.010 7 and 0.007 1, respectively. Finally, the authors used the MLR model to predict biomass for each pixel in the images of prediction set; images displayed in different colors for visualization based on pseudo-color images with the help of a Matlab program. The results show that using hyperspectral imaging technique to predict the biomass of Chlorella sp. and Spirulina sp. were better, but for the Isochrysis galbana visualization needs to be further improved. This research set the basis for rapidly detecting the growth of microalgae and using the microalgae as the bio-energy.