Background: Wnt signaling plays an important role in development and maintenance of many organs and tissues. The most-studied secreted Wnt inhibitors are sclerostin (SOST), Dickkopf-related protein 1 (DKK-1), and secreted frizzled related protein 1 (SFRP-1) which play important roles in bone turnover. The present study investigated the relationship between serum Wnt inhibitors and diseases with excessive ossification structures, such as ossification of posterior longitudinal ligament (OPLL), ankylosing spondylitis (AS), diffuse idiopathic skeletal hyperostosis (DISH), and ossification of yellow ligament (OYL). Methods: Twenty-five patients with AS, DISH, OPLL, or OYL were recruited in this study. Fasting peripheral blood samples were collected from all patients and nine controls. Various biomarkers of bone turnover including osteocalcin (OSC), osteoprotegerin (OPG), SFRP-1, DKK-1, and SOST were investigated. Results: Our data showed that serum levels of OSC were higher, but Dkk-1 levels were lower in AS, DISH, OPLL, and OYL patients than those in the controls. Serum levels of SFRP-1 were significantly higher in DISH patients than those in the controls. Serum levels of SOST were significantly higher in DISH and OPLL patients than both levels in the controls. Serum levels of OPG were lower in AS patients than those in the controls. Serum levels of OSC were higher in the OPLL patients than those in the AS patients. Serum levels of DKK-1, SFRP-1, SOST, and OPG were not significantly different between the different disease groups. Conclusions: In this exploratory study, both OSC and DKK-1 levels are correlated with the clinical conditions associated with excessive ossification, indicating that blood OSC and DKK-1 levels may serve as diagnostic biomarkers for AS, DISH, OPLL, and OYL. These findings may also help discover potential drug therapies for management of these diseases in the future.