The flow direction of microfluidics in biological applications is not limited to two dimensions, but often extends to three dimensions. Currently there are optical methods available for the measurement of 3-D microfluidic flow vectors, but with low spatial resolution. Line scan fluorescence correlation spectroscopy (FCS) was proposed to determine flow directions in 2-D within microchannels and small blood vessels in our previous work. Importantly, its spatial resolution was demonstrated to be as good as 0.5 microm. In this work, we extend line scan FCS to the third dimension for the characterization of 3-D flow velocity vectors. The spatial resolution is close to the diffraction limit using a scan length of 0.5 microm in all three dimensions. The feasibility of line scan FCS for 3-D microfluidic flow is verified by measurements in microchannels and small blood vessels of zebrafish embryos.