Single-turn extraction from the Japan Atomic Energy Agency AVF cyclotron with a K number of 110 using a flat-top (FT) acceleration system has been achieved to reduce the energy spread of an ion beam for microbeam formation with energy up to hundreds of MeV and to increase extraction efficiency from the cyclotron. In order to generate a FT waveform voltage using the fifth-harmonic frequency on a dee electrode, a FT resonator was designed using MAFIA code to achieve downsizing and low power consumption. The FT resonator, coupled to the main resonator through a coupling capacitor, covered the full range of the fifth harmonic frequency from 55 to 110 MHz. Various ion beams, accelerated using different acceleration harmonic modes of h=1 and 2, such as 220 MeV (12)C(5+) (h=2), 260 MeV (20)Ne(7+) (h=2), and 45 MeV H(+) (h=1), were developed by FT acceleration. A clear turn separation of the beam bunches was successfully observed at the extraction region of the large-scale AVF cyclotron with number of revolutions greater than 200. As a result, high extraction efficiency (over 95%) from the cyclotron was achieved. Single-turn extraction was confirmed by counting the number of beam bunches out of the cyclotron for an injected beam pulsed by a beam chopping system in the injection line. The energy spread of the 260 MeV (20)Ne(7+) beam was measured using an analyzing magnet, and we verified a reduction in the energy spread from DeltaE/E=0.1% to 0.05% by single-turn extraction after FT acceleration.