Janus amphiphilic particles have gained much attention for their important application value in areas as diverse as interfacial modification, sensors, drug delivery, optics, and actuators. In this work, we prepared Janus amphiphilic nanosheets composed of nitrogen-doped stratiform meso-macroporous carbons (NMC) and molybdenum sulfide (MoS2) for hydrophilic and hydrophobic sides, respectively. The dicyandiamide and glucose were used as precursors for synthesizing two-dimensional nitrogen-doped meso-macroporous carbons, and the molybdate could be anchored by the functional groups on the surface of carbon layers and then transform into uniformly MoS2 to form the Janus amphiphilic layer by layer NMC/MoS2 support. Transmission electron microscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, and Fourier transform infrared spectroscopy are used to demonstrate the successful preparation of Janus materials. As the typical interfacial enzyme, Candida rugosa lipase (CRL) immobilized on the Janus amphiphilic NMC/MoS2 support brought forth to improvement of its performance because the Janus nanosheets can be easily attached on the oil–aqueous interface for better catalytic activity (interfacial activation of lipases). The obtained immobilized lipase (NMC/MoS2@CRL) exhibited satisfactory lipase loading (193.1 mg protein per g), specific hydrolytic activity (95.76 U g–1), thermostability (at 55 °C, 84% of the initial activity remained after 210 min), pH flexibility, and recyclability (60% of the initial activity remained after nine runs). In terms of its application, the esterification rate of using NMC/MoS2@CRL (75%) is higher than those of NMC@CRL (20%) and MoS2@CRL (11.8%) in the “oil–water” biphase and CRL as well as NMC/MoS2@CRL in the one-phase. Comparing with the free CRL, NMC@CRL, and MoS2@CRL, the Janus amphiphilic NMC/MoS2 served as a carrier that exhibited more optimal performance and practicability.

An AIE dual-reactive supramolecular probe has been devised to simultaneously measure endogenous lipase and α-amylase activity in a homogeneous system. Fluorescence quantitative analysis of lipase and α-amylase in real biological samples enables rapid and accurate diagnosis of diseases.