In the present study, high-strength photoresponsive hydrogels were prepared by the photoinitiated copolymerization of acrylamide (AAm, hydrophilic hydrogen bonding monomer), 2-vinyl-4,6-diamino-1,3,5-triazine (VDT, hydrophobic hydrogen bonding monomer), and spiropyran-containing monomer (SPAA) in the presence of cross-linker poly(ethylene glycol) diacrylate (PEG575DA, Mn = 575). The double hydrogen bondings from AAm-AAm and diaminotriazine-diaminotriazine contributed to the considerable enhancement in tensile and compressive properties of the hydrogels, which showed an excellent ability to resist a variety of external forces. Fifteen minutes of UV (365 nm) irradiation led to the detachment of adhered cells due to the increased surface hydrophilicity caused by the isomerization of spiropyran moieties. Furthermore, repeated attachment/detachment of cells was realized by the alternate illumination of visible and UV light. Reverse gene transfection was carried out successfully by anchoring the PVDT/pDNA complex nanoparticles on the gel surface through hydrogen bonding between diaminotriazine motifs prior to cell seeding. Importantly, fibronectin (FN) modification combined with supplementing PVDT/pDNA complex nanoparticles after the first cycle of reverse gene transfection, so-called sandwich gene transfection, further increased the gene transfection level. A short time of UV light exposure could result in the nonharmful detachment of gene-modified cells from the gel surface. This high-strength photosensitive hydrogel holds potential as a reusable soft-wet platform for cell harvesting as well as gene transfection operation at higher efficiency.