We present the determination of the ground-state hyperfine splitting (νHFS) in laser-cooled 171Yb+ ions using our microwave quantum frequency standard (QFS). Employing Ramsey spectroscopy in a closed-loop measurement configuration, we have achieved a νHFS value of 12 642 812 118 469.0(8) mHz with a fractional uncertainty of 6.6×10−14. This result aligns with previously reported values and represents the highest accuracy reported to date for such measurements conducted in a single-shot closed-loop configuration, without averaging over multiple dates. The development of this accurate 171Yb+ microwave QFS holds promise as a transportable time-frequency reference for satellite navigation systems.