It is a challenge to non-invasively visualize in vivo the neovascularization in a three-dimensional (3D) scaffold with high spatial resolution and deep penetration depth. Here we used photoacoustic microscopy (PAM) to chronically monitor neovascularization in an inverse opal scaffold implanted in a mouse model for up to six weeks. The neovasculature was observed to develop gradually in the same mouse. These blood vessels not only grew on top of the implanted scaffold but also penetrated into the scaffold. The PAM system offered a lateral resolution of ∼45 μm and a penetration depth of ∼3 mm into the scaffold/tissue construct. By using the 3D PAM data, we further quantified the vessel area as a function of time.