A centrifugal force-based microfluidic device for a multiplexed analysis and an analyzing method using the same are provided. The microfluidic device includes a platform and a microfluidic structure including a plurality of chambers formed within the platform and valves positioned between the chambers. The microfluidic structure includes a sample separation chamber connected to a sample injection hole and a plurality of reaction chambers accommodating two or more types of markers specifically reacting to different types of target materials, separately by type.