In this article, the bias stress instability (BSI) of multilayered MoS₂ field-effect transistors (m-MoS₂ FETs) encapsulated in hydrophobic polymers [cyclized transparent optical polymer (CYTOP)] was systematically investigated under the pulse-mode operation. Compared with the dc mode, the threshold voltage shift (Δ Vth) under positive-bias stress was inversely larger than that for negative-bias stress. BSI in the m-MoS₂ FETs with CYTOP was negligibly affected by the external oxygen/moisture. Shallow hole traps and electron detrapping effects were strongly identified as the main causes of bias-polarity dependence on Δ Vth in the pulse mode. This can potentially be attributed to the sulfur vacancies in the m-MoS₂, which have a short trap lifetime of hundreds of microseconds. During the pulse-mode operation, various parameters, such as duty cycle, timeframe, bias polarity, and electric field strength, were systematically adjusted and experimentally applied to elucidate the origins of the asymmetry of the BSI in pulse mode. Atomic- and energy-band models, as well as recovery time constant extraction, are suggested to support the key mechanism of bias-polarity effects on BSI, which are, nicely, consistent with previous reports.