This paper describes an in vivo measurement technique to calculate dielectric properties of blood excluding the contributions from surrounding tissues, based on analysis of the pulsatile component bioimpedance spectroscopy data. A tetrapolar electrode system applied on the fingers is simulated by a pspice equivalent circuit, and current is injected with frequency ranging from 5 kHz to 100 MHz. The finger is modeled as three parts: arterial blood, surrounding tissues and incremental infused arterial blood during systole. The pulsatile curve is fitted to a Cole-Cole model by Levenberg-Marquardt (LM) nonlinear curve fitting. Using the pulsatile impedance data, electrical properties of blood and surrounding tissues can be separated successfully. Parameter accuracies are illustrated according to various noise levels and sampling rates. Keywords—Bioimpedance spectroscopy; pulsatile impedance wave; Cole-Cole model; nonlinear curve fitting.