Nitrate nitrogen (NO3-) and ammonium N (NH4+) are the two main forms of nitrogen (N). The effects of the NO3-:NH4+ ratio (0:100, 25:75, 50:50, 75:25, and 100:0) in the nutrient solution on growth, pigment content, photosynthetic parameters, chlorophyll (Chl) precursor content, and chloroplast mitochondrial ultrastructure of lettuce leaves were evaluated to determine the appropriate N ratio for the growth of lettuce. The results showed that a ratio of nitrate to ammonium of 75:25 compared with the single N form resulted in significantly higher fresh weights in the shoots and the lower parts of the lettuce. The growth rate, as indicated by the CO2 response curve, was increased because of the carboxylation efficiency and dark respiration rate (Rday) of the lettuce seedlings, and the difference between the light compensation point and CO2 compensation point was reduced under single nitrate N treatment. The contents of delta-aminolevulinic acid (ALA), porphobilinogen, uroporphyrinogen III, protoporphyrin IX, and Mg-protoporphyrin IX in seedlings at 75:25 nitrate:ammonium were higher than those in seedlings under the other treatments. The contents of Chl a and b were also the highest when the ratio of nitrate ammonium was 75:25. The chloroplast structure of lettuce leaves was intact, the cell membrane was completely closed to the cell wall, and the organelles were closely arranged on the cell membrane. The chloroplast was spindle-shaped and full of vesicles. The whole mitochondria were rich, the base particles were rich, the base granules were clear and rich, and the mitochondria exhibited the overall best state with a complete structure, rich sputum, and dense inclusions. The above results show that the nutrient solution with a ratio of nitrate to ammonium of 75:25 was more conducive to Chl synthesis and the promotion of photosynthesis and root growth in lettuce seedlings than the other treatments. The single nitrate N nutrient solution reduced Chl synthesis and photosynthesis in lettuce seedlings, while the single ammonium N nutrient solution affected the growth of lettuce seedlings, resulting in lower growth of lettuce shoots and roots. Therefore, the nutrient solution ratio of nitrate to ammonium of 75:25 is the most suitable for the lettuce seedling growth.