Envenoming by snakes from Lachesis genus is characterized by local pain, haemorrhage, oedema, mionecrosis, coagulation disturbs, hypotension and even shock. Shock is the lethal action of this venom and it is followed by an irreversible hypotension. Serine proteinases with kallikrein-like activity may act synergistically with other toxins, such as bradykinin potentiating peptides, causing this decrease in blood pressure level. There are few studies on the isolation of this snake venom serine proteinase class. The present study isolated a serine proteinase from Lachesis muta rhombeata venom through fast liquid protein chromatography. This enzyme showed high activity on chromogenic substrate for plasma kallikrein and lower on chromogenic substrate for plasmin and streptokinase-activated plasminogen. Also, this serine proteinase showed activity on bovine fibrinogen. Its proteolytic activity was also evaluated at the presence of possible inhibitors (EDTA, PMSF, benzamidine and 1,10-phenantroline) and only PMSF and benzamidine were able to decrease its enzymatic activity, confirming that it is a serine protease. The molar mass of pure enzyme was determined by MALDI/TOF mass spectrometry as 28.18 kDa. Additionally, the 55 first amino acid residues from N-terminal were sequenced by Edman degradation showing it is a novel enzyme similar to other snake venom serine proteinases deposited in data bank In conclusion, this study revealed a novel serine proteinase obtained from Lachesis muta rhombeata venom with possible kallikrein-like activity.