Biodiesel has been used more and more widely as it is environmentally friendly and sustainable. However, the traditional method of producing biodiesel often suffers from the low yield and high energy consumption. In order to improve the production efficiency of biodiesel, the ultrasound-assisted method was successfully applied to field pennycress (Thlaspi arvense L.) seed oil extraction and biodiesel transesterification in this study. The Box–Behnken design based on response surface methodology was used to optimize the multivariate processes of ultrasound-assisted extraction and transesterification. The optimal extraction conditions were: liquid to solid ratio of 25:1 mL/g, sonication time of 13 min, sonication amplitude of 90%, and extraction temperature of 45 ℃, with the maximal oil yield of 23.31 ± 0.51%. The optimal transesterification conditions were: methanol to oil ratio of 9.50:1 g/g, sonication amplitude of 90%, temperature of 60 °C, catalyst concentration of 1.36 wt%, and sonication time of 2.50 h, with the maximal biodiesel yield of 95.91 ± 0.28%. The seed oil obtained by ultrasound-assisted method and Soxhlet extraction method showed similar yield, fatty acid composition, and thermal stability, but the former had lower acid value, peroxide value, and free fatty acid content, which could be used as a raw material for the production of biodiesel more suitably. Almost all properties of biodiesel produced by ultrasound-assisted transesterification were compatible with those of biodiesel standards. Moreover, the energy consumption of ultrasound-assisted oil extraction and biodiesel production decreased by 94.84% and 55.50%, compared with that of the corresponding traditional procedures, respectively. Overall, ultrasound-assisted extraction of field pennycress seed oil and transesterification to produce biodiesel are efficient and environmentally friendly.