Next-generation high-performance biological and chemical sensors based on the emerging multitudinous two-dimensional (2D) layered materials have been attracting great attention in recent years. The performance of 2D biochemical sensors is strongly dependent on the structural defects, which provide indispensable active sites for sensitive and selective adsorption of analytes. However, achieving controllable defect engineering is still a big challenge. In the present work, we propose achieving superior biochemical sensor performance with high-surface-density grain boundaries (GBs), a kind of ubiquitous structural defects, in polycrystalline 2D thin films, which can be controllably synthesized. As a proof-of-concept, by utilizing the high-density GBs in monolayer (1L) WS2 films, we fabricated a series of surface plasmon resonance (SPR) sensors for mercury ion (Hg2+) detection. Our investigation has demonstrated substantial sensitivity enhancement of Hg2+ detection down to trace attomolar-level quantification (detection limit of 1 aM), which is ascribed to the abundance of active sites on high-density GBs. This work provides a promising avenue for the design of ultra-sensitive sensors toward commercialized products based on the GB-rich 2D layered materials.