The present work reports development of advanced nanosensor platform based on hierarchical 3D nanotubes assembled from MoS2 mono layers (3D MoS2NTs) for rapid and selective screening of well known carbamate fungicide, diethofencarb. The assembly of ultrathin MoS2 layers possessed mesopores and large specific surface area which enabled its potential implementation in electrode fabrication. Preparation of 3D MoS2NTs helped to avoid restacking and aggregation during its utilization as sensor material and manifested great sensing abilities towards electro-oxidation of diethofencarb. The LOD and LOQ for diethofencarb at 3D MoS2NTs/GCE were calculated as 0.32 ng μL−1 and 1.09 ng μL−1 in the concentration range of 1–55 ng μL−1 by square wave voltammetry (SWV) and further 0.63 ng μL−1 and 2.11 ng μL−1 in the concentration range of 1–35 ng μL−1 by differential pulse voltammetry (DPV). The developed 3D MoS2NTs nanosensor was successfully used to quantify diethofencarb in river water sample with satisfied recovery of 94.23%–105.46% justifying its analytical utility in pesticide screening. The study drives immense motivation towards sensing of hazardous pesticides using nanomaterials based advanced sensing devices for environmental monitoring.