In this work, we investigated the electrical characteristics of pentacene-based ambipolar organic thin-film transistors (OTFTs) by modifying the channel length. We fabricated a top contact device structure with sliver as the source and drain electrodes and heavy doped p-type silicon wafer as the gate electrode. The channel length of the pentacene-based ambipolar OTFTs are 50, 100, 250, and 400 μm; the channel width is fixed. The output current of the n-channel and p-channel decreases with increasing channel length. The saturated mobility and threshold voltage of both channels increase with the increase in channel length. The increase rate of saturated mobility and threshold voltage of the n-channel is larger than that of the p-channel. The influence of channel length on the electrical properties of the p-channel and n-channel is different. We utilized the gated-transfer length method to study the contact resistance between sliver and pentacene and the channel resistance of pentacene. Contact and channel resistance decrease with the increase in gate voltage in the saturation region. The total resistance of pentacene-based ambipolar OTFTs increases with channel length at a fixed gate voltage. However, n-channel total resistance has stronger gate voltage and channel length dependence than p-channel total resistance. This result reveals that electron transport in the device channel requires a larger driving voltage than in the hole. Selecting a suitable channel length is critical to obtain a well-balanced performance of the dual carriers that transport ambipolar OTFTs and to avoid a large loss in injection barrier.