For the development of a global navigation satellite system (GNSS), the third generation of BeiDou Navigation Satellite System (BDS-3) achieved full constellation for worldwide service on 23 June 2020. The new signals, B1C and B2a of BDS-3, further enhance the compatibility and interoperability between different GNSSs. In this study, we first assessed the quality of all the signals in BDS-3/GPS/Galileo. Then, to achieve the interoperability among BDS-3/GPS/Galileo, the inter-system bias (ISB), which appears if an inter-system difference exists between two GNSSs, was estimated at overlapping frequencies. Finally, we used the estimated ISBs in real-time kinematic (RTK) positioning. The results show the higher quality of the overlapping frequency B2a/L5/E5a than B1C/L1/E1 in terms of pseudo range multipath. The ISBs are stable both in the short term for one day and in the long term for over a year, which fit a zero-mean normal distribution well when the identical type of receiver is applied. Thus, it is reasonable to ignore the ISBs in the inter-system differences. With the estimated ISBs, the inter-system double-difference RTK can be achieved, which is called a tightly combined model (TCM) RTK. Compared with the traditional intra-system double-difference RTK, which is called a loosely combined model (LCM) RTK, the TCM RTK can achieve a higher success rate (SR) in terms of ambiguity resolution and higher positioning accuracy. In addition, the higher the cutoff elevation angle set, the greater the promotion can be obtained in SR. Even with a cutoff elevation angle of 50°, the SR of TCM is over 80%. Thus, it is important to apply TCM RTK when the observation conditions are limited, such as in dense jungles or the urban canyons.