The study aims to develop novel clinical immunohistochemical biomarkers for distinguishing small hepatocellular carcinoma (sHCC) from dysplastic nodules (DN). iTRAQ-2DLC-ESI-MS/MS technique was used to screen immunohistochemical biomarkers between precancerous lesions (liver cirrhosis and DN) and sHCC. A total of 1951 proteins were quantified, including 52 proteins upregulated in sHCC and 95 proteins downregulated in sHCC by at least 1.25- or 0.8-fold at p < 0.05. The selected biomarker candidates were further verified using Western blotting and immunohistochemistry. Furthermore, receiver operation characteristics (ROC) curves and logistic regression model were carried out to evaluate the diagnostic values of the biomarkers. Finally, aminoacylase-1 (ACY1) and sequestosome-1 (SQSTM1) were chosen as novel candidate biomarkers for distinction of sHCC from DN. A constructed logistic regression model included ACY1, SQSTM1, and CD34. The sensitivity and specificity of this model for distinguishing sHCC from DN was 96.1% and 96.7%. In conclusion, ACY1 and SQSTM1 were identified as novel immunohistochemical biomarkers distinguishing sHCC from DN. In conclusion, expression levels of CD34, ACY1, and SQSTM1 can be used to establish an accurate diagnostic model for distinction of sHCC from DN.