Cervical cancer caused mainly by high risk human papillomavirus (HPV) 16 and 18 strains is the second most prevalent cancer among women in Kenya. It is often diagnosed late when treatment is difficult due to very low percentage of women attending screening thus, mortalities remain high. The most available tests in low-and-middle-income countries (LMICs) have relatively low specificity, low sensitivity, require a laboratory setting and huge technical and financial support not readily available. HPV 16/18 E6 oncoprotein has been identified as a potential biomarker in a more specific early diagnosis of cervical cancer. This retrospective cross-sectional study developed a paper-based nanokit with enhanced detection of HPV 16/18 E6 oncoprotein for cervical cancer screening. The HRP labelled antibodies HPV 16 E6/18 E6-HRP (CP15) passively conjugated to citrate stabilized 20 nm gold nanoparticles were evaluated for immune sensing mechanism using a recombinant viral HPV E6 protein. The diagnostic accuracy was evaluated using 50 tissue lysates from formalin fixed paraffin embedded cervical biopsy, including control (n=10), Mild Dysplasia (n=10), Cervical intraepithelial neoplasia 3 (CIN3) (n=10), Cervical intraepithelial neoplasia 4 (CIN4) (n=10) and invasive carcinoma (n=10). The molecular technique used was dot blot molecular assay. A positive result was generated by catalytic oxidation of peroxidase enzyme on 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate. The gold nanoparticles were used to enhance the signal produced by peroxidase activity of horseradish peroxidase (HRP) enzyme giving a more sensitive assay as compared to use of non-conjugated antibody. This study provides a significantly high and reliable diagnostic accuracy for precancerous and cancerous lesions with a sensitivity of 90%, a specificity of 90%, a likelihood ratio for positive and negative tests as 9:1 and 1:9 respectively, a Positive Predictive Value of 97.3% and a Negative Predictive Value of 69.2%. The Limit of Detection obtained in the study was 0.0005 pg/ml. This study avails a sensitive, rapid test using paper-based nanotechnology which can be utilised in community-based screening outreaches particularly in low- and middle-income countries.