The automated fare collection (AFC) system has gained increasing popularity among transit systems worldwide. The AFC system is usually an entry-only system that only records the serial number of the smart card and the transaction time of each use. Neither the AFC data nor the bus global positioning system (GPS) could reveal the passenger’s alighting information, namely, alighting time and station. Hence, the station-to-station origin-destination (OD) trip information cannot be obtained directly from the available data sources. To address this problem, this paper proposes a methodology that estimates the OD matrix by using smart card and GPS data. In this paper, the characteristics of the basic data sources are first analyzed, based on which the bus arrival time is generated using the density-based clustering algorithm and a time correction strategy, based on which the passenger’s boarding station is identified. The alighting stations are inferred based on the characteristics of bus trip chaining, which could identify over 80% of the alighting stations on average. Finally, the proposed methodology is verified by a comprehensive field survey in Suzhou, China, with 100% sample rate.