The objective is to develop a novel methodology to analyze cortisol levels in saliva as an index of neuroendocrine response. In order to realize a portable, rapid and hand held biosensor of cortisol, we proposed an immuno-chromatographic test-strip based biosensor consisting of a disposable test-strip and a monitor. A fabricated disposable type test-strip has a size of 5 × 1.5 × 50 mm3. In order to realize an immuno-chromatographic test-strip, a glucose oxidase (GOD) -cortisol conjugate was newly synthesized, because the speed and selectivity of immunological reactions is particularly attractive as a strategy for molecular recognition of cortisol. The synthesized molecule was identified as the GOD-cortisol conjugate by both its molecular weight and specific detection of cortisol. Three min after addition of GOD enzyme assay solution, a color change reaction at a test line was observed. The R2 value for the calibration curve of the cortisol test strip was 0.95, and the relationship between its reflectance, r, and the cortisol level was r = 0.629 (1-0.232e (-1.78 x cortisol)). The fabricated cortisol biosensor enabled us to analyze cortisol concentrations between 1 - 10 ng/ml within 25 min of dropping a cortisol solution on the test strip. Thus, it is suggested that the cortisol biosensor may possibly be used for point-of-use measurements in hospitals.