Biomass, a renewable hydrocarbon, is one of the favorable sources of advanced carbon materials owing to its abundant resources and diverse molecular structures. Biomass-based two-dimensional carbon nanomaterials (2D-BC) have attracted extensive attention due to their tunable structures and properties, and have been widely used in the design and fabrication of electrochemical sensing platforms. This review embarks on the thermal conversion process of biomass from different sources and the synthesis strategy of 2D-BC materials. The affinity between 2D-BC structure and properties is emphasized. The recent progress in 2D-BC-based electrochemical sensors for health and environmental monitoring is also presented. Finally, the challenges and future development directions related to such materials are proposed in order to promote their further application in the field of electrochemical sensing.