The attachment and detachment of target cancer cells from homogeneous and binary mixtures in antibody-functionalized microchannels have been studied experimentally. Under the same intermediate flow rate, the attachment rate was found to be higher, and detachment flow rate was lower, for cell lines expressing the target receptor at a higher level. For cells that do not express the target receptor, the attachment rate was much lower but did not diminish, due to non-specific binding, and the detachment rate was much higher. The bio-functional microfluidic system performance in selectively isolating target cells from binary mixtures is quantitatively characterized. While the system sensitivity is typically very high, almost 100%, the specificity is lower than 90%. Applying a unique flow scheme of a slow flow rate, for maximum capture of target cells, followed by a faster flow rate, for maximum removal of non-target cells, the specificity is enhanced to levels above 95%, even for mixtures with target cells present at 11,000 relative concentration ratio.