In this article, we presented an ultrahigh-sensitivity creatinine (CRE) sensor based on the optical fiber surface plasmon resonance (SPR) effect and layer-by-layer self-assembly technology. The self-assembly technology is used to immobilize different polymer films containing creatininase (CA) molecules on the sensor surface. We found and verified that CRE decomposition products can induce the expansion effect of multilayer self-assembled polymer films and further cause the rapid reduction of the refractive index (RI) of polymer films. The SPR effect can detect this RI change and therefore infer the presence and concentration of CRE. The CRE detection results of SPR sensors with and without the self-assembly structure confirmed that the CRE sensitivity of SPR sensors improved by 11.6-fold and the limit of detection (LOD) reached $10^{-5}$ mg/mL when the SPR sensor combined with the self-assembly structure. Moreover, the selective recognition ability of the SPR sensor with the self-assembly structure has been confirmed by comparing the detection results of CRE and non-CRE samples. In addition, the underlying principle of the sensitization technique using self-assembled polymer films and specific biorecognition molecules (like CRE in this case) has the potential to be adapted for the detection of various other biomolecules, which could provide a new way for the development of highly sensitive and specific biosensors.