Zebrafish is an important model organism in biological research. One of the least explored tissues of zebrafish is blood, because the existing methods for isolating blood from this organism are tedious and irreproducible. The small volume of blood collected by these methods also prohibits many biochemical and cytological analyses. This technical obstacle limits the utilization of zebrafish in many applications, particularly in hematological research and plasma biomarker discovery. To overcome this limitation, we have established a novel method of extracting blood from zebrafish, based on the use of low centrifugal force to collect blood from a wound. This method consistently recovers more blood than traditional methods. Gel electrophoresis and flow cytometry showed that composition of blood harvested by this method is indistinguishable from traditional methods. The increase in yield enables us to perform biochemical experiments on zebrafish blood. In particular, we have demonstrated that quantitative proteomics can be performed on plasma collected from single zebrafish. Here, we have compared, by using shotgun proteomic analysis, the plasma proteomes of adult male and female zebrafish. Twenty-seven gender-dependent plasma proteins are identified and their biochemical importance discussed. Taken together, this novel technique enables analyses that were previously difficult to perform on zebrafish blood.