Blood lactic acid concentration is an important indicator for physiological functions. To develop a rapid and sensitive measurement technique for monitoring blood lactic acid may provide a useful tool in clinical diagnosis. We proposed to develop a microdialysis surface-enhanced Raman spectroscopy (microdialysis-SERS) approach to filter/reduce interference from other large metabolites in blood and enhance the detection sensitivity for blood lactic acid. In this study, a microdialysis probe was constructed using 13 kDa cut-off dialysis membrane. The dialysate was mixed with 50 nm Ag colloidal nanoparticles automatically in a micro-fluid chamber for SERS detection under blood microdialysis of Sprague-Dawley rat. The linear range of SERS-lactic acid measurement is 10 -5~3 x10 -4 M with R 2 value of 0.99. The optimal mixing flow rate of nanoparticles is 18 µl/min under microdialysis at constant flow rate (2 µl/min). Real time lactic acid monitoring in vivo also has been demonstrated using microdialysis-SERS system.