Polyanions are important sensing targets because of their wide variety of biological activities. We report a novel polyanion-selective fluorescence sensing system composed of a hybrid material of supramolecular hydrogel, enzymes, and aminoethyl-modified MCM41-type mesoporous silica particles (NH2-MCM41) encapsulating anionic fluorescent dyes. The rational combination of the polyanion-exchange ability of NH2-MCM41 and semi-wet supramolecular hydrogel matrix successfully produced three distinct domains; namely, cationic nanopores, hydrophobic nano/microfibers, and aqueous bulk gel phase, which are orthogonal to each other. The coupling of anion-selective probe release from NH2-MCM41 with translocation of the probe facilitated by enzymatic reaction enabled fluorescence resonance energy transfer-type sensing in the hybrid materials for polyanions such as heparin, chondroitin sulfate, sucrose octasulfate, and so forth. The enzymatic dephosphorylation catalyzed by phosphatase (alkaline phosphatase or acid phosphatase) that is embedded in gel matrix with retention of activity also contributed to improving the sensing selectivity toward polysulfates relative to polyphosphates. It is clear that the orthogonal domain formation of these materials and maintaining the mobility of the fluorescent dyes between the three domains are crucial for the rapid and convenient sensing provided by this system.