The aim of this study is to assess the ability of serum MMP-3 and serum CTX-II levels to differentiate between normal and early knee osteoarthritis case (eKOA). Subjects with clinical features of primary knee osteoarthritis of K-L Grade I and K-L Grade II with ages more than 45 years were included in the case group (98), and healthy adults with ages less than 40 years were included in the control group (80). Those having knee pain for the last 3 months but having no radiological features were labeled as K-L grade I and those having minimal osteophytes on radiographs were labeled as K-L Grade II. Antero-posterior views of knee and serum levels of MMP-3 and CTX II were estimated. Cases show significantly higher values of both biomarkers than in controls (p < 0.0001). Both biomarkers show significantly higher values with an increase in K-L Grades, that is, K-L Grade 0 versus I (MMP-3:​ p = 0.003;​ CTX-II:​ p = 0.002), K-L Grade I versus II (MMP-3:​ p < 0.000;​ CTX-II:​ p < 0.000). Multivariate analysis shows the dependence of both biomarkers only on K-L Grades. ROC analysis suggests cutoff value between KL Grade 0 and Grade I (MMP-3:​ 12.25 ng/mL;​ CTX II:​ 407.50 pg/mL) and between K L Grade I and Grade II (MMP-3:​ 18.37 ng/mL;​ CTX II:​ 528.00 pg/mL). The discriminating ability of CTX II is superior between normal population and eKOA (CTX II:​ Accuracy:​ 66.83%, p = 0.0002;​ MMP-3:​ Accuracy:​ 50.39%, p = 0.138), but MMP-3 is superior than CTX II between eKOA and mild KOA (CTX II:​67.52%, p < 0.000;​ MMP-3:​ 70.69%, p < 0.000).