By measuring the stem and leaf biomass, as well as the carbon (C), nitrogen (N), and phosphorus (P) contents in alfalfa, the effects of mowing frequency (three and four cuts, designated as C3 and C4, respectively) and fertilization level [the N:P2O5:K₂O application rates (kg/hm2) were 0:0:0(CK), 8.775:22.5:37.5(F1), 17.55:45:75(F2), 26.325:67.5:112.5(F3), and 35:90:150(F4), respectively] on organ biomass and the C, N, P stoichiometric characteristics of stems and leaves in the Horqin Sandy Land were investigated. The goal was to provide theoretical support for implementing high-yield cultivation practices in this region. The results showed that mowing frequency and fertilization level significantly affected alfalfa biomass and the C, N, P contents and stoichiometric characteristics in various organs (P<0.05). Under the condition of three mowings, aboveground biomass initially increased and then decreased with increasing fertilization levels, reaching a maximum value (1825.50 g/m2) under the F2 treatment, which was significantly higher than that under CK, F3, and F4 treatments (P<0.05). Under the condition of four mowings, aboveground biomass exhibited a ‘bimodal’ pattern, with the F1 and F3 treatments performing best. The C, N, P contents and stoichiometric ratios in different organs varied significantly among treatments. The highest carbon contents in new leaves, old leaves, reproductive stems, and vegetative stems were observed under treatments C3F1, C3F4, C3F2, and C3F2, respectively; the highest nitrogen contents were found under treatments C3F3, C3F1, C3F2, and C3F2, respectively; and the highest phosphorus contents were recorded under treatments C3F3, C3F1, C4F3, and C4F2, respectively. Within each organ, N and P contents were positively correlated, while stoichiometric ratios were mainly inversely regulated by the corresponding element contents, with C:N and C:P ratios negatively correlated with N and P contents. Comprehensive evaluation using the membership function method indicated that among the ten treatments, C3F2 had the highest comprehensive evaluation value (0.78), demonstrating the best effect on total biomass accumulation and element uptake in alfalfa.