Soil macronutrient nutrients (N, P and K) are critical for crop growth and agricultural production. The rapid and quantitative determination of soil nutrient content is of great importance to guide precise fertilization. To address the difficulty of synchronizing the detection of soil N, P and K with high sensitivity, this paper proposes a novel C4D microfluidic device with integrated 3D microelectrodes, which achieves lower detection limits for nutrients detection. The chip consisted of a crossover-type electrophoretic microchannels system and a 3D microelectrodes C4D system. 3D microelectrodes were constructed through clever design with only a single photolithography process required. The proposed microfluidic chips were used to detect potassium, ammonium, nitrate, and phosphate, with experimental results of standard solutions showing low detection limits of 5.24 × 10−5 g/L, 2.81 × 10−5 g/L, 2.35 × 10−5 g/L and 2.38 × 10−5 g/L. The microfluidic chips showed good reproducibility with relative standard deviations of less than 5 %. Separation experiments were also performed on soil samples with a resolution of K+ and NH4+ greater than 1.1. Accuracy was evaluated by adding 10 mg/L and 50 mg/L standards to the soil samples and results showed that recoveries were maintained at 80–120 %. The microfluidic chip with integrated 3D microelectrodes proposed in this work would effectively address the need for on-site, rapid detection of soil nutrients.