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 with different types of target materials, separately by type. At least one of the target materials is a standard material, and at least one of the markers is a standard marker specifically reacting with the standard material.