In order to achieve rapid, convenient and efficient soil nutrient determination in soil testing and fertilizer recommendation, a portable optical-fiber-sensor-based spectrophotometer including immersed fiber sensor, flat field holographic concave grating, and diode array detector was developed for soil non-metallic nutrient determination. According to national standard of ultraviolet and visible spectrophotometer with JJG 178-2007, the wavelength accuracy and repeatability, baseline stability, transmittance accuracy and repeatability measured by the prototype instrument were satisfied with the national standard of III level; minimum spectral bandwidth, noise and excursion, and stray light were satisfied with the national standard of IV level. Significant linear relationships with slope of closing to 1 were found between the soil available nutrient contents including soil nitrate nitrogen, ammonia nitrogen, available phosphorus, available sulfur, available boron, and organic matter measured by the prototype instrument compared with that measured by two commercial single-beam-based and dual-beam-based spectrophotometers. No significant differences were revealed from the above comparison data. Therefore, the optical-fiber-sensor-based spectrophotometer can be used for rapid soil non-metallic nutrient determination with a high accuracy.