Phosphorus (P) deficiency severely limits maize growth and yield, yet early detection remains challenging, as visible symptoms appear only after prolonged starvation. This study evaluated the capability of hyperspectral imaging (HSI) combined with machine learning to detect P deficiency in maize seedlings at both symptomatic and pre-symptomatic stages. Two greenhouse experiments were conducted: a long-term pot system under high and low P conditions and a short-term hydroponic experiment with three P concentrations of 500, 100, and 0 μmol/L phosphate (Pi). After long-term P deficiency, significant reductions in shoot biomass and Pi content were observed, while root biomass increased and nutrient profiles were altered. Hyperspectral signatures revealed distinct wavelength-specific differences across visible, red-edge, and near-infrared (NIR) regions, with P-deficient leaves showing lower reflectance in green and NIR regions but higher reflectance in the red band. A multilayer perceptron machine learning model achieved 99.65% accuracy in discriminating between P treatments. In the short-term experiment, P deficiency significantly reduced tissue Pi content within one week without affecting pigment composition or photosynthetic parameters. Despite the absence of visible symptoms, hyperspectral measurements detected subtle spectral changes, particularly in older leaves, enabling classification accuracies of 80.71–84.56% in the first week and 85.88–90.98% in the second week of P treatment. Conventional vegetation indices showed weak correlations with Pi content and failed to detect early P deficiency. These findings demonstrate that HSI combined with machine learning can effectively detect P deficiency before visible symptoms emerge, offering a non-destructive, rapid diagnostic tool for precision nutrient management in maize production systems.