Abstract: With the widespread application of novel nanomaterials, graphene oxide (GO) has inevitably entered agricultural ecosystems. Agricultural production takes place in open environments and is highly susceptible to the effects of such environmental nanomaterials. Current research on the effects of GO on crops is mostly limited to hydroponic conditions, failing to reflect real-world soil agriculture scenarios. To fill this gap and ensure agricultural safety, this study aims to investigate the effects of GO on tomato plants and soil under soil cultivation conditions and to determine the optimal concentration of GO suitable for tomato cultivation. The experiment used GO, soil collected from the potato onion germplasm nursery of Northeast Agricultural University, and the tomato variety "Dongnong 722" as test materials. Different concentrations of GO (50 mg/kg, 100 mg/kg, and 200 mg/kg) were added to the soil, with soil without GO (0 mg/kg) serving as a control. Tomato seedlings at the two-leaf-one-heart stage were transplanted into 9 cm × 9 cm seedling pots. Samples were collected before transplanting (day 0), 20 and 40 days after transplanting to measure relevant indicators of tomato growth, nutrient uptake, soil chemical properties, and enzyme activity. The results showed that GO exhibited a hormonal effect on tomato growth: low to moderate concentrations promoted growth, while high concentrations had no significant promoting effect. The growth of tomato was optimal at a concentration of 100 mg/kg: plant height and stem diameter at 40 days after transplanting were significantly higher than the control and the 200 mg/kg treatment; total plant dry weight and root dry weight were significantly increased at both 20 and 40 days; root volume and root surface area also increased significantly, especially at 40 days. Regarding nutrient uptake, the nitrogen content at 40 days was significantly higher in the 100 mg/kg treatment than in all other treatments, and the phosphorus content at both sampling time points was significantly higher in 100 mg/kg treatment than in the control and the 200 mg/kg treatment. Only 100 mg/kg treatment significantly increased the potassium content of tomato plants. Regarding soil properties, GO had no significant effect on pH. Electrical conductivity increased with increasing GO concentration at 20 days, but showed the opposite trend at 40 days. At both 20 and 40 days, the ammonium nitrogen content in the 100 mg/kg treatment was significantly higher than in other treatments, while nitrate nitrogen content showed the opposite trend. At 20 days, the 100 mg/kg treatment had the highest available phosphorus content, and at both 20 and 40 days, the 100 mg/kg treatment had the highest available potassium content. Regarding enzyme activity, the 100 mg/kg treatment showed the highest urease activity, acid phosphatase activity increased with increasing GO concentration, and catalase activity showed no significant difference among all treatments. In conclusion, the optimal GO concentration for tomato growth in soil is 100 mg/kg. This concentration promotes tomato growth by regulating the activity of key soil enzymes, optimizing the ratio of ammonium nitrogen to nitrate nitrogen, reducing phosphorus and potassium loss, and improving root development and nutrient absorption. This study fills a knowledge gap in research on the agricultural effects of GO under soil cultivation conditions, confirms the positive regulatory effect of appropriate amounts of GO on tomatoes under soil cultivation conditions, and provides theoretical support and a safe concentration reference for the application of GO in tomato cultivation.