Background and Aims <p>Manure affects soil N content and composition, thereby influencing the nutritional quality of crop grains. This study aimed to assess the effects of long-term manure application on maize and soybean grain quality, as well as soil amino acids pools within a crop rotation system.</p> Methods <p>Four treatments were selected from a maize-maize-soybean rotational fertilisation field experiment that has been ongoing since 1979: no fertiliser (CK), mineral fertiliser (NPK), low-rate manure (13.5 t ha⁻¹) with NPK (M1NPK), high-rate manure (27 t ha⁻¹) with NPK (M2NPK). Soil amino acids pools, as well as protein and amino acids content of grains was evaluated.</p> Results <p>The results indicate that soil organic nitrogen fraction increased with high-rate manure additions. Long-term high-rate manure application significantly increased the levels of free amino acids while reducing the proportion of soil alkaline hydrolysable amino acids. Maize grain protein content increased with higher manure addition rates; however, albumin and globulin content peaked at 0.76 and 0.45&#xa0;g 100&#xa0;g⁻¹, respectively, under M1NPK. Manure addition had no significant effect on the protein content or its fractions in soybean grains. Soybean amino acids content peaked 348&#xa0;mg&#xa0;kg⁻¹ under M2NPK. A strong positive correlation was observed between the protein content of both maize and soybean grains and the soil's total N and amino acids pool.</p> Conclusions <p>Long-term manure application in a crop rotation system increased soil amino acids pool and enhanced crop grain protein and amino acids content; however, lower manure rate (13.5 t ha⁻¹) additions was more favourable for maize albumin and globulin.</p>