Abstract: The aim of this study was to reveal nitrogen (N) preference and optimal ammonium-nitrate ratio for growth of sugarcane (Saccharum officinarum L.). A hydroponic experiment was conducted with cultivar ‘Yunzhe05-51’ grown under a total N supply of 10 mmol/L, with seven ammonium-nitrate ratios, i.e., 0∶100, 20∶80, 40∶60, 50∶50, 60∶40, 80∶20, and 100∶0. Plant growth and physiological responses in relation to N assimilation were characterized. Results showed that the plant biomass of ‘Yunzhe05-51’ peaked at an ammonium-nitrate ratio of 20∶80. Under this treatment, plants showed the best root morphology, highest N assimilation-related enzyme activity, and relatively higher N uptake and utilization efficiency. Root growth began to display depression when the ammonium-nitrate ratio reached 40∶60, while shoot growth was significantly limited when the ammonium-nitrate ratio increased to 80∶20. Plants under single ammonium supply had the highest levels of N concentration, N uptake per unit root length, free amino acid content, and soluble protein content, but showed lower N assimilation-related enzyme activity and the lowest N utilization efficiency, as well as the most stunted plant growth and root morphology. In conclusion, exploring the combined effects of ammonium and nitrate on plant growth, N assimilation and utilization were better than single administration of either ammonium or nitrate, and the optimal ammonium-nitrate ratio for ‘Yunzhe05-51’ was 20∶80.