Amorphous organic room-temperature phosphorescent (RTP) materials are promising for their facile preparation and processability, while the conformation effects of phosphors at amorphous state are lack of study in comparison with the rigid effects due to the commonly irregular assembling and dispersal of phosphors in rigid systems. Herein, we report a series of phosphorescent molecules modified by polyhydroxy galactose, whose RTP emission at the amorphous state can be regulated by controlling the conformational distortion of the phosphorescent segments. Further, a strong RTP emission is facilely obtained by the co-assembling between polyhydroxy phosphors and polyhydroxy matrices (α-CD, β-CD, and chitosan). Owing to the rigid effect of the enhanced hydrogen bonding cross-linking, the highest RTP quantum yield reaches 19.4%; whereas, the RTP emissions of assemblies become conformation insensitive. The conflicting relationship between the conformation effect and rigid effect is attributed to the differences between aggregated single-component systems and dispersed assembling systems. Besides, the unique and different moisture responsiveness of the co-assembling samples is discovered and further applied in data encryption. The research expands the scope for designing amorphous pure organic RTP materials with supramolecular strategies and shows a modularized approach for assembling-enhanced phosphorescence.