Stimulus‐responsive large‐scale cell culture microcarriers are promising biological materials for large‐scale cell production. Herein, multifunctional thermal and magnetic microcarriers (TMMs) made of poly(N‐isopropylacrylamide) (PNIPAM), polylysines (PLLs), gelatin methacryloyl (GelMA), poly(ethylene glycol) diacrylate (PEGDA), N,N‐methylenebis(acrylamide) (MBA), Fe3O4 and SiO2 are prepared to achieve large‐scale enzyme‐free cell harvesting and also can be used as cells stereogrowth scaffold to mimic cells suspension state to develop novel application, such as recombinant proteins produced. In TMMs, NIPAM and other materials are mixed and then underwent in situ radical polymerization by UV irradition with 2‐hydroxy‐2‐methylpropiophenone as initiator, in which GelMA provides cell adhesion sites and PLLs enable a positive microenvironment with SiO2 as filler. As enzyme‐free cell microcarriers, cell harvesting from TMMs by PNIPAM are achieved at 25 °C through vortex oscillation within 15 min and TMMs can be recycled at least for four times by magnetic recovery. Moreover, TMMs can be used as stereoculture scaffold to let cells in suspension as adherent state to improve gene transfection efficiency. Combined with magnetothermal effect, TMMs can further improve gene transfer to obtain industrialization of proteins expression. Therefore, TMMs are potential for the development of cell manufacturing and the expansion of cell stereogrowth application for recombinant proteins production.