Special functionalization of semiconducting polymer dots (Pdots) is highly desired to expand their applications in chemo/biosening. Herein, carboxyl-functionalized poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1',3}-thiadiazole)] dots covalently tagged with aminated beta-cyclodextrin (NH2-CD) have been designed to construct a ratiometric sensor for cholesterol (Cho). Using CD-Pdots as energy donors with rhodamine B (RB) as energy acceptors, a fluorescence resonance energy transfer (FRET) pair has been built because the host guest interaction between RB and CD attached to Pdots brings donors and acceptors into close proximity. In the presence of Cho, the acceptors will depart from the donors because of the competitive inclusion interaction between Cho and RB with CD, resulting in the hindering of the FRET process between CD-Pdots and RB. On the basis of the turn-on fluorescence of CD-Pdots and turn-off fluorescence of RB, a sensitive ratiometric method for the determination of Cho in the concentration range from 25 to 350 nM with a detection limit of 4.9 nM was achieved. The proposed method was validated to determine free Cho in human serum samples with satisfactory results.