Amphiphilic biomembrane structures determine significant biological functions and are extensively used as structure models to learn from and study nature. Many biomimetic amphiphilic membranes have been established to connect natural and artificial substances. In this paper, taking advantage of the intercalation and assembly properties of the layered double hydroxides (LDHs), the amphiphilic LDHs/arachidic acid (AA) nanocomposite Langmuir-Blodgett (LB) ultrathin films (UTFs) were fabricated by the LB technology. The CdTe quantum dots (QDs) were incorporated into the LB monolayers via a layer-by-layer (LbL) method based on the electrostatic interaction between LDHs and CdTe QDs. The amphiphilic (CdTe QDs@LDHs/AA)(n) nanocomposite LB UTFs were composed of CdTe QDs@LDH5 hydrophilic segments and hydrophobic layers formed by the long alkyl chain of AA. Because of the spacing effect of amphiphilic AA, the fluorescence intensity of CdTe QDs was enhanced about 10-fold, and the fluorescence lifetimes (38.96 ns vs 17.63 ns) and quantum yield (QY %) (17.56 vs 5.96) have been improved compared to that of the counterpart by the LbL method. The fluorescence intensity of CdTe QDs increased by about fivefolds in the presence of LDHs compared with the counterpart without LDHs, which can be attributed to the two-dimensional confinement effect of LDHs. The amphiphilic nanocomposite LB UTFs were used to detect volatile organic compounds (VOCs) with various polarities. The amphiphilic nanocomposite LB UTFs exhibited two kinds of fluorescence response to VOCs: irreversible fluorescence quenching for amine VOCs with strong polarity and reversible fluorescence enhancement for non-amine VOCs. The fluorescence response mechanism was investigated and can be attributed to the amphiphilic structure of the LB UTFs and the selective adsorption of different VOC molecules. Therefore, this fluorescence quenching/enhancement dual-model response of amphiphilic nanocomposite LB UTFs can be applied into the selective detection of VOCs.