Vitamin D insufficiency is closely related to various kinds of metabolic diseases. Acted as a marker of vitamin D status, 25-HydroxyvitaminD3 detection possesses important practical significance. In this study, highly sensitive fluorescent detection of 25-HydroxyvitaminD3 using truncated affinity-improved aptamers were developed, based on fluorescence intensity changes of PicoGreen (PG) generated upon binding to double-stranded DNA (dsDNA) formed through hybridization of aptamer and corresponding complementary strand. Four truncated aptamers were obtained by intercepting the small hairpin loop as the functional domain and retaining double helix structural domains of different lengths that exist in the selected original 25-HydroxyvitaminD3 aptamer. Under the optimized PG concentration, we conducted comparison experiments for affinity and specificity of these four truncated aptamers. Among them, the shortest aptamer with only 21 bp, D3-4, was found to show the highest affinity and specificity to 25-HydroxyvitaminD3, with the limit of detection of 0.04μg/mL, which solved the problem that original long aptamer could not applied for this fluorescent detection of 25-HydroxyvitaminD3. The truncated 25-HydroxyvitaminD3-specific aptamer with highly enhanced affinity performs promising application in sensitive detection of 25-HydroxyvitaminD3.