Background <p>In Egypt, bladder cancer is the second most common cancer among men and the fourth most common cancer in the population. It is tightly associated with genetic alterations. Microtubule-associated protein 1 light chain 3B (LC3) is a unique marker of autophagosomes. Beclin-1 participates in the formation of the autophagosome by mediating the localization of other autophagy proteins to the membrane of the pre-autophagosome. Impaired autophagy is linked to the development of several diseases as cancer and autoimmunity. This work aims to investigate the significance of LC3 and beclin-1 immunohistochemical and gene expressions in bladder cancer diagnosis.</p> Methods <p>This study included seventy-four tissue samples of primary urothelial bladder cancer. The tissues were investigated for gene expression, as well as histopathological and immunohistochemical analysis. The genetic and immunohistochemical expressions were correlated with the clinicopathological data.</p> Results <p>In the cancerous tissues, there were significant upregulations of LC3 and beclin-1 mRNA and protein expression compared to the adjacent control tissues (<i>P</i> &lt; 0.001 for each). A non-significant positive correlation between <i>LC3 and beclin-1</i> expression was detected (r =0.212 , P<i>=0.21</i>). Receiver operating curve (ROC) results showed that <i>LC3</i> at a cut-off point of ≥ 4.3 could be a potential diagnostic marker of bladder cancer with a sensitivity of 96.2% and a specificity of 90.6% (P &lt; 0.001). In addition, <i>beclin-1</i> at a cut-off point ≥ 2.3 could be a predictor of bladder cancer with a sensitivity of 98% and a specificity of 89% (P &lt; 0.001).</p> Conclusion <p><i>LC3</i> and <i>beclin-1</i> expressions can be possible diagnostic markers of bladder cancer. Additionally, they are associated with tumor grade and stage.</p>