In this paper, a kind of multi-functional polylactic acid-based membrane with adjustable aggregate thickness and properties was prepared. When the number of assembled layers was 20 bilayers, the mean thickness of every bilayer was found to be 10.75±0.25 nm, and the tensile strength was increased by 20% compared with the polylactic acid (PLA) films. The assembled layered double hydroxides (LDHs) could serve as a type of promising organic-inorganic luminescent material, and endow PLA with photoluminescent properties. Besides, the photoluminescence of the composite films was discovered to be highly tunable by simply adjusting the structural constituents of LDHs, and the fluorescence intensity was uniformly enhanced as the number of assembled layers increases, which was not eliminated by the isolation due to cellulose nanocrystals (CNCs). Moreover, the composite films exhibited excellent fluorescence stability in comparison with the literature. After being repeatedly bleached 1000 times by confocal laser scanning fluorescence microscope, the fluorescence intensity of the PLA/(CNCs/Mg-Al-Eu LDHs) 10 film remained 96%. It could be concluded that the immobilization of CNCs and LDHs realized the microscopic controllability of the optical functional molecular spacing. With satisfactory fluorescence, the resulting PLA-based membranes exhibited considerable application potential in optoelectronic devices and anti-counterfeiting packaging.