Organophosphorus (OPs) compounds can endanger human health and the environment by inhibiting the acetylcholinesterase enzyme. But these compounds have been widely used as pesticides due to their effectiveness against all kinds of pests. In this study, a Needle Trap Device (NTD) packed with mesoporous organo-layered double hydroxide (organo-LDH) material and coupled with gas chromatography-mass spectrometry (GC-MS) was employed for the sampling and analysis of OPs compounds (diazinon, ethion, malathion, parathion, and fenitrothion). In this way, the [magnesium–zinc–aluminum] layered double hydroxide ([Mg–Zn–Al] LDH) modified with sodium dodecyl sulfate (SDS) as a surfactant was prepared and characterized by FT-IR, XRD, BET, and FE-SEM, EDS, and elemental mapping techniques. Then, various parameters such as relative humidity, sampling temperature, desorption time, and desorption temperature were evaluated by the mesoporous organo-LDH:NTD method. The optimal values of these parameters were determined using response surface methodology (RMS) and central composite design (CCD). The optimal temperature and relative humidity values were obtained as 20 °C and 25.0%, respectively. On the other hand, the desorption temperature and time values were in the range of 245.0–254.0 °C and 5 min, respectively. The limit of detection (LOD) and limit of quantification (LOQ) were reported in the range of 0.02–0.05 mg m−3and 0.09–0.18 mg m−3, respectively, which shows the high sensitivity of the proposed method compared to the usual methods. The repeatability and reproducibility of the proposed method (by calculating the relative standard deviation) was estimated in the range of 3.8–10.10 which indicates the acceptable precision of the organo-LDH:NTD method. Also, the desorption rate of the stored needles at 25 °C and 4 °C, was determined to be 86.0% and 96.0%, respectively after 6 days. The results of this study proved that the mesoporous organo-LDH:NTD method can be utilized as a fast, simple, environmentally friendly, and effective method for sampling and determining OPs compounds in the air.