Serine-proteases are hydrolases that act in diverses systems including central nervous system (CNS), i.e., neuropsin, u-PA and thrombin. Neuropsin, presented in neurons and glial cells, seems to be involved in learning and memorization process and may act in Alzheimer, epilepsy and multiple sclerosis diseases. The u-PA, a plasminogen activator, is presents in pathological process caused by inflammatory demyelination (i.e. multiple sclerosis). At last, thrombin is known as presenting a systemic effect of blood coagulation, however it was identify as acting in neurons synapses. In attempt to identify structural features responsible for their different biological functions in CNS, we made a comparative study between their structures. We also realized a study involving the proteases and their substrates with aim to discuss possible interaction. Neuropsin, thrombin and u-PA crystal structures were obtained from Protein Data Bank (PDB). As expected, the primary structure alignment revealed the conservation of catalytic triad (His57, Asp 102, Ser 195) and the S1 position (Asp189). However, variations in S2 and S3 positions were observed among neuropsin (Pro95 and, probably, Lys175), u-PA and thrombin (Gly216, Gly226).  Comparing the secondary structures, neuropsin and u-PA presented 2 β-sheet while thrombin presented 4 β-sheet. Interestingly, u-PA possesses 2 α-helix; neuropsin and thrombin presented the same α-helix arrangement of u-PA however the first one had an extra smaller one and the other one presented 2 more different α-helix. In order to stabilize their folding, three disulfide bonds are also strictly conserved in these enzymes. The electrostatic potential map analysis showed an equilibrium of negative and positive regions in neuropsin, u-PA and thrombin. The interaction analyses involving these enzymes and their substrates were done using Deepview/ Swiss-PDBview 3.7 program and showed several interactions between them. Our analysis suggested that specific difference of these enzymes are more related to the distribution of the amino acids in their surface than to their final 3D-structure folding. This study can contribute in the future for a more specific designing of new inhibitors for the treatment of diseases in which these enzymes are involved. Suported by: CNPq, UFF.