Knee osteoarthritis (KOA) is the most common osteoarthritis in lower limbs, and gait measurement is important to evaluate walking function of KOA patients before and after treatment. The third generation Intelligent Device for Energy Expenditure and Activity (IDEEA3) is a portable gait analysis system to evaluate gaits. This study is to evaluate the accuracy and reliability of IDEEA3 for gait measurement of KOA patients. Meanwhile, gait differences between KOA patients and healthy subjects are examined. Twelve healthy volunteers were recruited for measurement comparison of gait cycle (GC), cadence, step length, velocity and step counts between a motion analysis system and a high-speed camera (GoPro Hero3). Twenty-three KOA patients were recruited for measurement comparison of former five parameters between GoPro Hero3 and IDEEA3. Paired t-test, Concordance Correlation Coefficient (CCC) and Intraclass Correlation Coefficient (ICC) were used for data analysis. All p-values of paired t- tests for GC, cadence, step length and velocity were greater than 0.05 while all CCC and ICC results were above 0.95. The measurements of GC, cadence, step length, velocity and step counts by motion analysis system are highly consistent with the measurements by GoPro Hero3. The measurements of former parameters by GoPro Hero3 are not statistically different from the measurements by IDEEA3. IDEEA3 can be effectively used for the measurement of GC, cadence, step length, velocity and step counts in KOA patients. The KOA patients walk with longer GC, lower cadence, shorter step length and slower speed compared with healthy subjects in natural speed with flat shoes.