The incompatibility of most organic solvents with acetylcholinesterase (AChE) inhibition assay normally limits pesticide extraction efficiency in sample pretreatment, which might cause false negatives in real world sample assessment. Herein, a novel method has been developed for an improved AChE inhibition assay via organic solvent extraction combined spontaneous in situ solvent evaporation on microfluidic paper-based analytical devices. Enzyme pre-immobilization procedure was spared and AChE was added to the system after sampling step until a complete in-situ solvent evaporation process was performed on chip. IC levels of the six investigated organophosphate and carbamate pesticides indicated a completely eliminated influence of solvents on AChE behavior with the new method. Most importantly, analytical performances were significantly improved in food sample measurements. Reduction in matrix effect was observed when acetonitrile was adopted for lettuce sample pretreatment instead of water. Studies on different pesticides suggested a remarkably decreased discrimination effect on recoveries from sample pretreatment with the new developed method. The recovery level for phoxim spiked head lettuce samples reached (107.5 ± 14.2) %, in comparison with that of (18.6 ± 1.4) % from water-based extraction. Spiked water and apple juice samples with carbaryl concentration of as low as 0.02 mg L were also successfully recognized with the present method by visual detection. This is the first report on direct sampling of organic extracts for AChE inhibition assay on-chip and it might provide a new perspective for real world sample assessments involving bio-reagents.