Limited data are available on the effects of 5-aminolevulinic acid (ALA) on plant photosynthesis in relation to the nitrogen (N) level. In this study, we investigate photosynthetic responses to ALA in canola plants (Brassica napus L.). We used wild-type plants without ALA addition (controls), wild-type plants with exogenous ALA application, and transgenic plants that endogenously overproduced ALA. The plants were grown hydroponically in nutrient solutions with low, middle, and high concentrations of N. Our results indicate that plants in both treatment groups had higher chlorophyll contents and net photosynthetic rates and lower intracellular CO2 concentrations in the leaves, as compared to controls. Furthermore, simultaneous measurement of prompt chlorophyll fluorescence and modulated 820-nm reflections showed that the active photosystem II (PS II) reaction centers, electron transfer capacity, and photosystem I (PS I) activity were all higher in treated plants than controls at all N levels; however, the responses of some photochemical processes to ALA were significantly affected by the N level. For example, under low N conditions only, a negative ΔK peak appeared in the prompt chlorophyll fluorescence curve, indicating a protective effect of ALA on electron donation via activation of the oxygen-evolving complex. Taken together, our findings suggest that ALA contributes to the promotion of photosynthesis by regulating photosynthetic electron transport under various N levels. These findings may provide a new strategy for improving photosynthesis in crops grown in N-poor conditions or reduced N-fertilization requirements.