Measurement-device-independent quantum key distribution (MDI-QKD) is proved to be able to eliminate all potential detector side channel attacks. Combining with the reference frame independent (RFI) scheme, the complexity of practical system can be reduced because of the unnecessary alignment for reference frame. Here, based on polarization multiplexing, we propose a time-bin encoding structure, and experimentally demonstrate the RFI-MDI-QKD protocol. Thanks to this, two of the four Bell states can be distinguished, whereas only one is used to generate the secure key in previous RFI-MDI-QKD experiments. As far as we know, this is the first demonstration for RFI-MDI-QKD protocol with clock rate of 50 MHz and distance of more than hundred kilometers between legitimate parties Alice and Bob. In asymptotic case, we experimentally compare RFI-MDI-QKD protocol with the original MDI-QKD protocol at the transmission distance of 160 km, when the different misalignments of the reference frame are deployed. By considering observables and statistical fluctuations jointly, four-intensity decoy-state RFI-MDI-QKD protocol with biased bases is experimentally achieved at the transmission distance of 100km and 120km. The results show the robustness of our scheme, and the key rate of RFI-MDI-QKD can be improved obviously under a large misalignment of the reference frame.
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