Highly efficient enantioselective separation and quantitative recoveries of d- and l-tryptophan in aqueous and real samples can be achieved, with a monolithic molecularly imprinted polymeric fiber that serves both for micro-solid phase extraction and ultratrace sensing, without any false-positive (non-specific) contribution and cross-reactivity, in the range of 0.15–30.00ngmL −1 with detection limit as low as 0.0261ngmL −1 (relative standard deviation=0.64%, signal/noise=3). The proposed method combining molecularly imprinted micro-solid phase extraction fiber and a complementary molecularly imprinted polymer–carbon composite fiber sensor is proven to be useful for clinical diagnosis of stress-related diseases caused by acute tryptophan depletion.