As an ideal raw material for the production of astaxanthin, H. pluvialis was drawing attention during the last few years, there are some research topics initiated to find out the synthetic pathway of astaxanthin in H. pluvialis. In this study, confocal microscopic Raman technology was utilized to analyze the point-by-point mapping for H. pluvialis, and the visualization of pigment such as carotenoid and astaxanthin content were achieved. By comparing the Raman spectra of H. pluvialis and standard product of astaxanthin, and using the C = C stretching vibration of the Raman intensity as the main indicator for carotenoids, the visual spatial distribution for the carotenoids content was obtained. The MCR-ALS was applied to analyze the Raman data of H. pluvialis, and the information of astaxanthin was extracted to achieve real-time spatial distribution. The visualization of astaxanthin content shows that MCR-ALS is very effective for extracting the information of astaxanthin content from H. pluvialis. By exploring the spatial distribution of carotenoids and astaxanthin contents, analyzing the spatial distribution rules during its growth, Raman spectroscopy technology can be utilized to investigate the internal components of the pigment (ataxanthin, etc.) in H. pluvialis, which make it as an effective methodology to monitor the accumulation and changing mechanism of pigment content in microalgae.