Three-dimensional (3D) multicellular spheroids are a new generation in vitro cell model, however, their applications are severely limited by difficulties in their generation. Here patterned poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel films were synthesized for their generation. Instead of polymerization of HEMA monomers in the presence of a cross-linker, here the PHEMA films were synthesized by cross-linking furan-functionalized linear PHEMA, PHEMA-furan, and maleimide-functionalized linear PHEMA, PHEMA-mal, via Diels-Alder (DA) reaction between furan and maleimide groups. A thermal treatment temperature of 75 °C was chosen for the cross-linking reaction. The occurrence of DA reaction was confirmed by IR spectra. Using this method, cross-linked PHEMA films with smooth surface were successfully synthesized in situ in the well of cell culture plates. The films were then patterned by simply adding water to swell them. Highly ordered, honeycomb-like wrinkling patterns were successfully obtained by adjusting the furan and maleimide contents in the precursor linear polymers. The patterned hydrogel films were used to generate multicellular spheroids. Guided by the patterns, 3D spheroids with narrow size distribution, tunable size, and high cell viability were successfully obtained. The patterned PHEMA films reported here exhibited a lot of advantages. The patterning method was quite simple and required no template or special equipment. They were synthesized in situ in commercial cell culture plates. Particularly, thanks to the clean nature of the DA reaction, no low molecular weight monomer, cross-linker, initiator, or catalyst, which were potentially cytotoxic, was involved in the film synthesis, and no byproduct was produced and left in the film. The resulting films presented a high biocompatibility, allowing the avoidance of the tedious washing step. The films synthesized here were expected to have high potential for massive production of well-defined multicellular spheroids.