The increasing strength of new structural materials and the span of new structures, accompanied by aesthetic requirements for greater slenderness, are resulting in more applications of long-span structures. In this paper, serviceability control technology and its design theory are studied. First, a novel tuned mass damper (TMD) with controllable stiffness is developed. Second, methods for modeling human-induced loads are proposed, including standing up, walking, jumping and running, and an analysis method for long-span floor response is proposed based on a finite element model. Third, a design method for long-span floors installed with a multiple TMD (MTMD) system considering human comfort is introduced, largely based on a study of existing literature. Finally, a design, analysis and field test is conducted using several large scale buildings in China including the Beijing Olympic Park National Conference Center, Changsha New Railway Station and the Xi’an Northern Railway Station. The analytical and field test results show that the MTMD system designed using the proposed method is capable of effectively mitigating the vertical vibration of long-span floor structures. The study presented in this paper provides an important reference for the analysis of vibration serviceability of similar long-span floors and design of control system for these structures.