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A more proper and realistic multilayer dielectric model of spherical biological cell, in which nuclear was taken into consideration, was proposed based on the classic dielectric model in this paper. The general analytical method was also deduced and analyzed in detail in calculating the time courses of transmembrane potentials of both inner and outer membranes induced by constant and time-varying electric field. The time course of transmembrane potential of the outer membrane for multilayer dielectric model was compared to that of the classical model. It is shown that the latter is larger than the former, particularly for a cell with larger nuclear. The time courses of transmembrane potentials of both inner and outer membranes induced by pulsed electric fields (PEFs) with different durations were also studied based on the multilayer dielectric model. Long PEF targets outer membrane mainly, and there is little influence to cell nucleus, mitochondrion, and other organelles; thus, it causes electroporation to the outer membrane. As the pulse duration decreases, the electroporation effect changes gradually from the outer membrane to intracellular organelle membrane. Ultrashort PEF (tens of nanoseconds) induces larger voltage across the inner membrane and acts mostly on intracellular substructures. However, submicrosecond PEF (several hundreds of nanoseconds) can induce significant voltages across both the inner and outer membranes, therefore, causing damage to both the inner and outer membranes. This property of submicrosecond PEF has much practical value for tumor treatment.