This paper introduces the chemiluminescence (CL) detection in oscillating reaction-based determinations using the analyte pulse perturbation technique, a straightforward and expeditious approach to deriving quantitative analytical information from oscillating chemical reactions. The behavior of the H2O2–KSCN–CuSO4-NaOH oscillating system in the presence of luminol was examined by using the proposed detection method and the classical potentiometric technique. Some analytical and practical aspects of both detection systems are discussed. A new analytical method for the determination of vitamin B6 based on the sequential perturbation produced by different amounts of this substance on the oscillating chemical system was also developed in order to assess the potential of CL detection for routine analyses. The calibration curve thus obtained was linear over the range 0.5–2.0 μmol of vitamin B6, and the precision and throughput were also quite good (3.04% as RSD and nine samples h−1, respectively). The proposed method was validated by determining the vitamin in pharmaceutical preparations.