Rapid quantitative immuno-detection of haptens by the lateral flow assay without “typical” competitive inhibition results is studied. In the present study, we describe an immuno-threshold-based assay for the quantification of cortisol. It gives a signal which is directly proportional to the cortisol concentration in plasma samples with a performance time of only 5 min. This technique provides a practical calibration curve with detection limit of 3.5 ng/ml. The precision of the assay is 6% (intra-assay coefficient of variation, CV) and 10% (inter-assay CV). Cross-reactivity with related steroids is acceptably low: corticosterone (3.38%), cortisone (2.08%), deoxycorticosterone (2.00%), 17α-hydroxyprogesterone (0.39%), and progesterone (0.05%). Furthermore, the test strips show the advantages of long storage time and high stability that allow mass production and preparation of large batches. A one-step cortisol whole blood test derived from this plasma lateral flow assay has then been performed. It is a rapid chromatographic immunoassay designed for quantitative determination of cortisol in whole blood samples. It requires no sample pretreatment and gives result within 15 min. In principle, with this rapid and sensitive immunoassay, the immuno-test strips can be employed for detecting all low-molecular-weight haptens. It may also be a useful and convenient dipstick format for drug detection.