Acetylcholinesterase (AChE) is an important enzyme in the control of the neuronal action potential and sensitive to organophosphate inhibition. Brain fish AChE is less sensitive to organophosphate inhibition than AChE from terrestrial animals, although this sensitivity is variable among species and has not yet been fully evaluated in fish species. In this setting, inhibition kinetic constants for progressive irreversible inhibition of brain acetylcholinesterase due to methyl-paraoxon exposure were determined in three fish species (Mugil liza, Genidens genidens and Lagocephalus laevigatus) and hen (Gallus domesticus). Enzyme extraction using a detergent was shown to be adequate, and samples presented activity inhibition in high substrate concentrations and suppression of inhibition by methyl-paraoxon in the presence of the substrate, similar to kinetic patterns from purified enzyme preparations. Catfish (G. genidens) AChE presented the highest sensitivity among the evaluated fish species (IC50 = 1031.20 nM ± 63.17) in comparison to M. liza and L. laevigatus (IC50: 2878.83 ± 421.94 and 2842.5 ± 144.63 nM respectively). The lower dissociation constant (Kd = 20.3 ± 2.95 μM) of catfish AChE showed greater enzyme affinity for methyl-paraoxon, explaining this species higher sensitivity to organophosphates. Hen AChE presented higher ki (900.57 ± 65.3 mM-1min-1) and, consequently, greater sensitivity to methyl-paraoxon, explained by a lower Kd (0.6 ± 0.13 μM). Furthermore, hen AChE did not differentiate between the propionylthiocholine and acetylthiocholine substrates, indicating easier access of methyl-paraoxon to the hen enzyme activity site. The results obtained herein indicate a suitable extraction of AChE and, despite different inhibition kinetic constants, demonstrate that fish AChE is less sensitive to methyl-paraoxon, probably due to reduced access to the catalytic center which provides greater enzyme substrate selectivity.