Primary investigation of Platymonas subcordiformis was performed with laser scanning microscopic technology. Both autofluorescence spectra and autofluorescence imaging of Platymonas subcordiformis were achieved by one photon excitation of 488 nm Ar+ laser. Autofluorescence images revealed cup-shaped object inside the Platymonas subcordiformis cell, and the corresponding fluorescence peak located at 682 nm was attributed to chloroplast. Moreover, in single channel mode, there existed a round-shaped object in chloroplast center, showing strong fluorescence by 800 nm femtosecond laser excitation. Also, the authors obtained both the individual cup-shaped chloroplast and round-shaped object image and the overlaid images by using dual-channel mode. Meanwhile, six main fluorescence peaks were found. Single photon excitation can be used to obtain autofluorescence image and autofluorescence spectra of Platymonas subcordiformis, while two photon excitation combined with multitrack mode and Lambda mode can be used not only to observe internal structure, but also for the analysis of existence of biological and chemical substances with higher sensitivity than single photon excitation. LSCM technique can be a promising tool for rapid, convenient, real-time and effective investigation in ocean algae.