•3D-nanostructured MoS2 is grown using metal-organic chemical vapor deposition.•The 3D-nanostructured MoS2 (3DN-MoS2) is rolled via an ethanol drop-casting process.•The MoS2 nanoscroll self-assembles in the direction of high strain energy.•The scrolling of 3DN-MoS2 leads to opened active sites in nanostructure interlayers.•This technology enables low concentration toxic gas detection at room temperature. Assembly operations can transform simple structures into complicated topologies such as crumples, folds, and scrolls, offering extraordinary electronic and optical properties. Two-dimensional (2D) transition metal dichalcogenides (TMDC) nanoscrolls have attracted attention for their distinctive and excellent characteristics. However, their chemically inert surface limits scalability in various applications. This study exploits the scrolling characteristics of three-dimensional nanostructured 2D MoS2 (3DN-MoS2) to improve NO2 detection. By growing 3DN-MoS2 and applying high strain energy through an ethanol solution-based drop-casting process, the material self-assembles in the direction of high strain energy. To confirm the applicability of opened active sites in nanostructure interlayers, we fabricated a scrolled 3DN-MoS2 gas sensor, demonstrating a 28-fold sensitivity enhancement compared to pristine 3DN-MoS2. The unique assembly operation of nanostructures enables versatile applications, including the detection of toxic gases at low concentrations and low operating temperatures.