The photosynthetic performances of regenerated protoplasts of Bryopsis hypnoides, which were incubated in seawater for 1, 6, 12, and 24 h, were studied using chlorophyll (Chl) fluorescence and oxygen measurements. Results showed that for the regenerated protoplasts, the pigment content, the ratios of photosynthetic rate to respiration rate, the maximal photosystem II (PSII) quantum yield (F v /F m ), and the effective PSII quantum yield (Φ PSII ) decreased gradually along with the regeneration progress, indicated that during 24 h of regeneration there was a remarkable reduction in PSII activity of those newly formed protoplasts. We assumed that during the cultivation progress the regenerated protoplasts had different photosynthetic vigor, with only some of them able to germinate and develop into mature thalli. The above results only reflected the photosynthetic features of the regenerated protoplasts at each time point as a whole, rather than the actual photosynthetic activity of individual aggregations. Further investigation suggested a relationship between the size of regenerated protoplasts and their viability. The results showed that the middle-sized group (diameter 20-60 µm) retained the largest number of protoplasts for 24 h of growth. The changes in F v /F m and Φ PSII of the four groups of differently sized protoplasts (i.e. < 20, 20-60, 60-100, and > 100 µm) revealed that the protoplasts 20-60 µm in diameter had the highest potential activity of the photosynthetic light energy absorption and conversion for several hours.