This work demonstrated leukocyte count and two-part leukocyte differential from the whole blood based on a portable microflow cytometer system. Leukocytes are selectively stained with a fluorescent dye, Acridine Orange (AO). The blood sample is then pumped through a disposable microfluidic device for fluorescence sensing. Under blue LED excitation (460nm), the green fluorescence from DNA and the red fluorescence from RNA are detected simultaneously with two photomultiplier tubes (PMT). Leukocytes are counted and differentiated into two parts, lymphocyte versus non-lymphocyte, based on their fluorescence signatures. The results, including leukocyte absolute count and lymphocyte ratio, matched well with the commercial hemacytometer counts (maximal error 9.0%, correlation coefficient ∼0.8). We also tested the system for the first time under a zero-gravity (zero-G) environment that facilitated its application in space missions.