<p>Centrifugal microfluidics has emerged as a promising platform for automated bioanalysis, such as nucleic acid testing and immunoassays. In particular, real-time centrifugal microfluidic PCR provides a highly effective solution for point-of-care molecular diagnostics in resource-limited settings. However, during the development of these systems, monitoring their operational state for evaluation or calibration is difficult due to the inherent conflicts among thermal, optical, and centrifugal interactions. To address this challenge, this study presents a wireless, in-situ calibration ‘chip’ with multiple functions for system calibration and evaluation by being operated in a way similar to a normal disk chip. The calibration ‘chip’ consists of multiple different structural layers, e.g., sensing, optical, fluidic and electrical layers, integrating multiple functions including temperature calibration, fluorescence signal simulation, dynamic fluid monitoring, and mechanical sensing. To allow the in-situ calibration ‘chip’ to reasonably fit the centrifugation platform, it is powered by embedded rechargeable batteries and meanwhile Bluetooth-based wireless data transmission is adopted. Experimental results demonstrate that the calibration ‘chip’ is capable of performing multiple different tasks based on comprehensive sensing and actuation mechanisms, which is helpful to conveniently perform in-situ monitoring of the centrifugal microfluidic system with an on hand ‘tool’.</p>