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Cationic horseradish peroxidase (HRP) is usually used in сhemiluminescent enzyme immunoassay (CL-EIA), one of highly sensitive analytical methods. However, HRP has drawbacks connecting with quick decay of CL signal and, in some cases, insufficiently high sensitivity. We demonstrated that anionic plant peroxidase isolated from soybeans (SbP) is more effectively biocatalysts than HRP-C and is able to oxidize luminol in the absence of enhancers. In addition, unlike HRP-C, SbP produces a long-term CL signal. Although SbP is a more potent biocatalyst in luminol oxidation than HRP-C itself, in the presence of enhancers HRP-C produces a higher CL intensity than SbP. On the other hand, HRP-C enhancers do not practically increase SbP-induced CL. At screening of some phenothiazines we showed that 3-(10’-phenothiazinyl)propane-1-sulfonate (SPTZ) is a first potent enhancer of CL induced by anionic SbP. Also, the simultaneous introduction of SPTZ and 4-morpholinopyridine (MORP) in the substrate mixture resulted in an additional increase of CL as well as a decrease of the lower detection limit (LDL) of SbP. The SbP-catalyzed chemiluminescent signals in presence of SPTZ and SPTZ/MORP are long-term. At comparison of the three fully optimized systems, SbP–SPTZ– MORP versus HRP-C–SPTZ–MORP versus HRP-C–PIP, it demonstrated that the SbP system possessed significantly higher sensitivity and lower LDL value. The SbP-SPTZ-MORP system was successfully employed in CL-EIA for determination of human thyroglobulin, one of markers of thyroid gland cancer. These results open up very promising perspectives for using the SbP-SPTZ-MORP system in ultra-sensitive immunoassays.