Soil available phosphorus (AP) deficiency and shortage of phosphate rocks limit cotton production in China. Therefore, pool-culture experiments were conducted in 2019 and 2020 using two cotton cultivars (CCRI-79, low-P tolerant; SCRC-28, low-P sensitive) under three soil AP levels (P0: 3 ± 0.5, P1: 6 ± 0.5, and P2 (control): 15 ± 0.5 mg kg−1) to ascertain the effect of soil AP on boll biomass and lint yield. P0 and P1 decreased the P concentration and net photosynthetic rate (Pn) of subtending leaves, thus, reducing boll biomass and lint yield. Additionally, soil AP deficiency decreased boll wall:boll, lint:boll, and lint:seed, and increased seed:boll ratio. Upper fruiting branch positions (FB9–12) had higher lint:seed ratio and proportion of the total lint yield under low soil AP. Moreover, soil AP deficiency also reduced the sucrose transformation rate (Tr) and activities of sucrose-metabolizing enzymes, such as ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco), sucrose phosphate synthase (SPS), and sucrose synthase (SuSy), while increased carbohydrate levels (soluble sugar, sucrose, and starch) and the activity of cytosolic fructose-1,6-bisphosphatase (cy-FBPase) in the subtending leaves. The sucrose and starch contents, cy-FBPase, and SPS activities of SCRC-28 were more sensitive to low soil AP than CCRI-79. Higher Tr and activities of initial Rubisco and SuSy in the subtending leaves improved boll biomass and lint yield.