The synthesis of a series of β-cyclodextrin modified mesoporous silica nanoparticles with hydroxyl, amino, and thiol groups was described. A comparison of their mucoadhesive properties and potential as a drug delivery system for superficial bladder cancer therapy was made. The thiol-functionalized nanoparticles exhibit significantly higher mucoadhesivity on the urothelium as compared to the hydroxyl- and amino-functionalized nanoparticles. This is attributed to the formation of disulfide bonds between the thiol-functionalized nanoparticles and cysteine-rich subdomains of mucus glycoproteins. An anticancer drug, doxorubicin, was loaded into the mesopores of the thiol-functionalized nanoparticles, and sustained drug release triggered by acidic pH was achieved. The present study demonstrates that thiol-functionalized mesoporous silica nanoparticles are promising as a mucoadhesive and sustained drug delivery system for superficial bladder cancer therapy.