The development of queens of Apis cerana cerana in isolated conditions was investigated, and optimal preservation methods were explored. A bionic queen storage device was first fabricated using polylactic acid via 3D modeling and printing. Under controlled artificial conditions at 35°C, the survival duration of worker bees was measured across varying humidity levels, feeding regimens, and group sizes. Comparisons were further made between isolated and in-colony queens in terms of body weight, ovary mass, and spermatheca diameter. Results showed that, 65% relative humidity (RH) extended worker bees’ median survival time by 6 days compared to 55% RH. A diet consisting of solid sugar cubes, water, and pollen prolonged median survival by 14.5 days relative to diets of sucrose solution ( w sucrose=50%) with pollen. Worker groups of 70 individuals reached a median survival of 20.5 days, outperforming groups of 30 and 50 bees. When accompanied by at least 50 attendant workers in the bionic storage device, queens showed no significant differences in body mass, ovary mass, and spermatheca diameter compared to in-colony queens. Notably, groups with 70 accompanying workers, which exhibited longer median survival, effectively reduced the frequency of worker replenishment in the device. Collectively, optimal preservation of isolated queens was achieved under 65% RH with 70 accompany workers provided solid sugar cubes, water, and pollen. This established isolated preservation system enables batch storage of queens in a stable environment and supports improved efficiency in selective breeding programs.