Hyaluronidases (HAase) are involved in various physiological and pathological processes and have been reported as urinary marker for bladder cancer. In this study, a novel ratiometric fluorescent sensing system based on both aggregation-induced emission (AIE) and aggregation-induced quenching (ACQ) was developed to quantitatively assess hyaluronidase level. First, a tetraphenylethylene derivative with positive charges (TPE-2N(+), typical ALE molecule) at both ends and an anthracene derivative with positive charge at one end (AN-N+, typical ACQ molecule) was synthesized. These two positively charged compounds were then mixed with a negatively charged hyaluronan (HA), which induced the aggregation of the compounds as well as the nanoparticles formation as a result of electrostatic complexation, with TPE-2N(+) acting as cross-linking agent. The aggregation also caused the efficient quenching of the emission of AN-N+ due to ACQ effect, as well as the fluorescence enhancement of TPE-2N(+) due to ME effect. In the presence of HAase, the enzymatic reaction led to the degradation of and triggered disassembly of the nanoparticles; as a result, the emission of AN-N+ was restored and that of TPE-2N(+) was suppressed. This fluorescence variation affords the system a robust ratiometric biosensor for HAase, and the ratio of fluorescence intensity for AN-N+ (I-414) to that for TPE-2N(+) (I-474) can be used as the sensing signal for detecting HAase activity. In this system, hyaluronan serves not only as the scaffold for nanoparticle formation but also as the substrate for enzymatic reaction. This assay system is operable in aqueous media with very low detection limit of 0.0017 U/mL and is capable of detecting HAase in biological fluids such as serum and urine. This strategy may provide a new and effective approach for developing other enzyme assays.