To study the combined effects of multiple nitrogen (N) sources and salinity on the growth and physiology on macroalgae, we cultured Ulva prolifera under three N levels (N0, 0.1235 mg L-1; N1, 0.6 mg L-1; and N2, 4.4 mg L-1; the ratios were 18:74:8 for NH4-N, NO3-N, and NO2-N, respectively) and three salinity conditions (15, 25, and 35). Then, the growth, pigment content, photosynthetic performance, superoxide dismutase (SOD) activity, and contents of soluble protein and carbohydrates were measured. The results showed the following: (1) Compared to that grown at salinity 25, the growth of U. prolifera decreased under salinity 35, especially under the N0 and N2 levels, but there were no significant effects of salinity 15 under any of the N levels. (2) There were no significant effects of salinity on the chlorophyll a (Chla) content, but compared to the content at salinity 25, the chlorophyll b (Chlb) content was enhanced by salinity 15 and 35; lower ratio values between Chla and carotenoids (Car) occurred under the salinity 25 treatment. Under each salinity condition, the pigments were enhanced by a high N level. (3) A relatively higher salinity level decreased the photosynthetic oxygen evolution rate, while a higher N level increased this value. Compared to the rate at salinity 25, the dark respiration rate (Rd) significantly increased at salinity 15 under the N0 condition. (4) SOD activity was enhanced by a high N level, but no significant effects of salinity were observed. (5) The carbohydrate content was enhanced at salinity 35 under the N0 and N1 levels, and under salinity 15, this value increased with increasing N levels. In conclusion, although the growth of U. prolifera decreased at high N levels under high salinity conditions, a high N level induced an increase in photosynthesis, while no significant decrease in growth occurred. These findings indicate that low salinity and high N levels may be nonnegligible reasons why this species thrives, and low salinity was the better choice when this species was used for wastewater treatment.