Novel, comb-like polymeric surfactants with polyurethane backbone (PUSs) having alkyl chains of a carbon number from 4 to 18, and poly(ethylene glycol) (PEG) chains of 9 and 23 ethylene oxide (EO) units as the side chains were synthesized. Their aqueous solutions showed cloud points which were dependent on the PEG chain length rather than the alkyl chain length. However, no cloud point was observed below 100 °C for PUS having alkyl chains of a carbon number of 18 and PEG chains of 23 EO units. The interfacial properties of the PUSs such as surface activity and micelle-forming property in the aqueous system were characterized by surface tension measurement and fluorospectroscopic technique using pyrene as the probe, respectively. The surface-active properties of the PUSs were analyzed with respect to the effectiveness and the efficiency of adsorption at the air/water interface and the micellization characteristics. Surface activity of PUSs with PEG chains of 9 and 23 EO units showed maxima at carbon numbers of 8 and 12 for alkyl chains, respectively. Such tendencies were concluded to be mainly related to the variation in the effectiveness of adsorption with alkyl chain length, in addition to the efficiency of adsorption for PUS with PEG chain of 9 EO units.