Wall shear stress (WSS) is of fundamental physiological and pathological significance. Current measurement technologies suffer from poor spatial resolution or cannot measure instantaneous values in a label-free manner. Here we demonstrate dual-wavelength third-harmonic-generation (THG) line-scanning imaging, for instantaneous wall shear rate and WSS measurement in vivo. We used the soliton self-frequency shift to generate dual-wavelength femtosecond pulses. Simultaneous acquisition of dual-wavelength THG line-scanning signals extract blood flow velocities at adjacent radial positions for instantaneous wall shear rate and WSS measurement. Our results show the oscillating behavior of WSS in brain venules and arterioles at micron spatial resolution in a label-free manner.