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Abstract 3D organoid models have recently seen a boom in popularity, as they can better recapitulate the complexity of multicellular organs compared to other in vitro culture systems. However, organoids are difficult to image because of the limited penetration depth of high‐resolution microscopes and depth‐dependent light attenuation, which can limit the understanding of signal transduction pathways and characterization of intimate cell‐extracellular matrix (ECM) interactions. To overcome these challenges, phototransfer by allyl sulfide exchange‐expansion microscopy (PhASE‐ExM) is developed, enabling optical clearance and super‐resolution imaging of organoids and their ECM in 3D. PhASE‐ExM uses hydrogels prepared via photoinitiated polymerization, which is advantageous as it decouples monomer diffusion into thick organoid cultures from the hydrogel fabrication. Apart from compatibility with organoids cultured in Matrigel, PhASE‐ExM enables 3.25× expansion and super‐resolution imaging of organoids cultured in synthetic poly(ethylene glycol) (PEG) hydrogels crosslinked via allyl‐sulfide groups (PEG‐AlS) through simultaneous photopolymerization and radical‐mediated chain‐transfer reactions that complete in <70 s. Further, PEG‐AlS hydrogels can be in situ softened to promote organoid crypt formation, providing a super‐resolution imaging platform both for pre‐ and post‐differentiated organoids. Overall, PhASE‐ExM is a useful tool to decipher organoid behavior by enabling sub‐micrometer scale, 3D visualization of proteins and signal transduction pathways.