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In this paper, we successfully developed Lamb-wave sensors in a two-port delay line topology with suspending GaN thin films on silicon substrates. The liquid insusceptibility of the lowest order symmetric mode (S0) Lamb wave was experimentally investigated, and the relative frequency shift to water loading of the two-port S0-wave sensors was found to be as small as 0.12%. Our GaN-on-Si sensors, exhibiting a high mass sensitivity (272 cm2/g) and a sharp signal response at a low concentration (1 μg/mL) of anti-bovine serum albumin, were demonstrated to be suitable for chemical and biological sensing in liquid environments. In addition, the small size of the sensors and their potential to be integrated with a wide range of GaN-based devices allow for a robust, low-cost miniature sensing system to be manufactured with GaN-on-Si substrates.