Degradation is among the most important properties of biomaterial scaffolds, which are indispensable for regenerative medicine. The currently used method relies on the measurement of mass loss across different samples and cannot track the degradation of an individual scaffold in situ. Here we report, for the first time, the use of multiscale photoacoustic microscopy to non-invasively monitor the degradation of an individual scaffold. We could observe alterations to the morphology and structure of a scaffold at high spatial resolution and deep penetration, and more significantly, quantify the degradation of an individual scaffold as a function of time, both in vitro and in vivo. In addition, the remodeling of vasculature inside a scaffold can be visualized simultaneously using a dual-wavelength scanning mode in a label-free manner. This optoacoustic method can be used to monitor the degradation of individual scaffolds, offering a new approach to non-invasively analyze and quantify biomaterial–tissue interactions in conjunction with the assessment of in vivo vascular parameters.