Planktonic algae are single-celled organisms, and their photosynthetic activity responds sensitively to external stress, so they are good test organisms for the comprehensive toxicity test of water quality. Obtaining the response law of the photosynthetic activity state of the tested algae species to the main environmental factors such as temperature and light, and mastering the culture conditions that effectively control the photosynthetic activity state and concentration of the tested algae species are essential for the comprehensive toxicity detection of water quality. Taking the model test organism Chlorella pyrenoidosa as the research object, the changes of photosynthetic activity of Chlorella pyrenoidosa under different temperature gradients and light intensities were studied in this work. The results show that the changes of photosynthetic activity and concentration of the algae species under different gradient illuminations are very obvious. Under low light (75 μE, 125 μE), the average photosynthetic activity of algae species is about 0.60 while the concentration of algae species basically does not increase. Under medium light (175, 225, 300, 375 μE), the average photosynthetic activity of algae species is around 0.57 and the concentration of algae species has increased significantly at this time. Among them, the best lighting for the experiment is 375 μE. Under high light (475 μE and 600 μE), the average photosynthetic activity of the algae species is lower than 0.56 (initial activity), which can stress the growth state of the algae species. It is also shown that the photosynthetic activity and concentration of algae species also changes significantly under different gradient temperatures. At medium and low temperatures (5, 15, 25 °C), the average photosynthetic activity of algae species is about 0.59, the concentration of algae species increases with the increase of temperature, and the best temperature is 25 °C. Under high temperature (30, 35, 40 °C), the photosynthetic activity of the algae species declines rapidly until inactivation. It is indicated that the photosynthetic activity and growth rate of planktonic algae can be controlled by controlling light and temperature, which can provide a standard method for cultivating the tested algae samples for a online water quality comprehensive toxicity meter, thereby laying a foundation for the development of the portable water quality comprehensive toxicity meter.