Objective: To investigate the levels of a deoxynivalenol (DON) biomarker in the urine of subjects living in two China provinces with different geographic locations and dietary patterns, and estimate their dietary DON exposures and health risks. Methods: First morning urine samples were collected on three consecutive days from 599 healthy subjects—301 from Henan province and 298 from Sichuan province—to analyze the total DON concentrations (tDON) after β-glucuronidase hydrolysis using a high-performance liquid chromatography tandem mass spectrometry-based method. The consumption of cereal foods in the previous 24 h before each urine collection was recorded using a duplicate diet method. DON exposure levels were estimated based on the urinary tDON concentrations. Results: Total DON were detected in 100% and 92% of the urine samples from Henan and Sichuan, respectively. Mean urinary tDON concentrations were 52.83 ng/mL in Henan subjects and 12.99 ng/mL in Sichuan subjects, respectively. The tDON levels were significantly higher in the urine of Henan subjects than that of the Sichuan subjects (p < 0.001). Urinary tDON levels were significantly different among age groups in both areas (Henan: p < 0.001; Sichuan: p = 0.026) and were highest in adolescents aged 13–17 years, followed by children aged 7–12 years. Based on the DON biomarker and exposure conversion reported by the European Food Safety Authority (EFSA), the mean estimated dietary intakes of DON were 1.82 μg/kg bw/day in Henan subjects and 0.45 μg/kg bw/day in Sichuan subjects. A total of 56% of Henan subjects and 12% of Sichuan subjects were estimated to exceed the PMTDI of 1 μg/kg bw/day. Consistent with urinary tDON levels, the highest estimated dietary DON intakes were also in children and adolescents aged 7–17 years. For all kinds of wheat-based foods except dumplings, the consumptions were significantly higher in Henan than those in Sichuan. The mean consumption of steamed buns was 8.4-fold higher in Henan (70.67 g/d) than that in Sichuan (8.45 g/d). The mean consumption of noodles in Henan (273.91 g/d) was 3.6-fold higher than that in Sichuan (75.87 g/d). Conclusions: The levels of urinary DON biomarker and the estimated dietary DON intakes in Henan province were high and concerning, especially for children and adolescents. The overall exposure level of Sichuan inhabitants was low.