The discovery of Piezo channels has advanced the understanding of mechanobiology, which acts as mechanosensitive cation channels in sensing mechanical stimuli and regulating various physiological functions. Despite the electrophysiological current recording, localized Piezo activation measurement in cells still faces challenges. In this study, we developed a Piezo1-Ca2+ biosensor based on F?rster Resonance Energy Transfer (FRET) technology by fusing a calcium-sensing module closely to the channel pores, hence to dynamically detect Piezo1 activation. The biosensor expressed in 293T cells showed ～120% FRET change to the specific chemical agonist Yoda1, whose response to Piezo1 activation was evaluated with its key-point mutation (L1342G/L1345G). By comparing cytoplasmic and Lyn-tagged membrane calcium biosensors, Piezo1-Ca2+ FRET biosensor primarily reflects localized calcium signals near the plasma membrane. Furthermore, by combining Piezo1-Ca2+ FRET imaging with varying microfluidic shear conditions, the biosensor exhibited significant yet transient responses to shear stress (～70% FRET change at 2.8 dyn/cm²), with a threshold of effective activation around 1.0 dyn/cm² (i.e., 10 μN/cm²). In conclusion, the developed Piezo1-Ca2+ FRET biosensor demonstrated Piezo1 activation by chemical agonist and shear force, which provides an imaging tool with improved spatiotemporal resolution for elucidating the mechanosensitivity of Piezo1 channels. The local specificity for Piezo1-Ca2+ signal detection by the biosensor requires individual interpretations under different scenarios.