The synergistic effect of natural guar gum (GG), konjac gum (KGM) and sodium 2-oxopropanoic acid sodium (2-OAS) to designed a novel physical cross-linked three-dimensional network structure GG@2-OAS@KGM as a carrier of active microorganisms for mold and yeast sensitive detection. At the ratio of 6:2:2 (w/w/w), GG@2-OAS@KGM possessed a uniform porous structure. After treatment for 120 h, the hydrogel exhibits higher water holding capacity (WHC, 71.7 %) and swelling stability (SR, 667.5 %), and it also show a stable entrapment efficiency for chromogenic substrates as 95.7 % and 95.3 %, separately. Especially, the 2-OAS as a structural modifier of the free chromophore group indoxyl, to improve the surface rigidity of π-π conjugation effect to enhance the blue fluorescence characteristics of mold and yeast at 365 nm wavelength after incubated for 36 h. Compared with the national standard GB 4789.15-2016 agar plate method (≥5 d), the GG@2-OAS@KGM as active microorganisms carrier provided the fluorescent and visible detection methods can improve mold and yeast detection efficiency and reduce background interference of detection, obviously. This work provides a new reference for the microbial stimuli-responsive chromogenic hydrogel, with potential application in microbiological rapid detection method.