There have been significant advances in the field of nanofluidics, and novel technologies such as single-cell analysis have been demonstrated. Despite the evident advantages of nanofluidics, fluid control in nanochannels for complicated analyses is extremely difficult because the fluids are currently manipulated by maintaining the balance of driving pressure. To address this issue, the use of valves will be essential. Our group previously developed a nanochannel open/close valve utilizing glass deformation, but this has not yet been integrated into nanofluidic devices for analytical applications. In the present study, a nanofluidic analytical system integrated with multiple nanochannel open/close valves was developed. This system consists of eight pneumatic pumps, seven nanochannel open/close valves combined with piezoelectric actuators, and an ultra-high sensitivity detector for non-fluorescent molecules. For simultaneous actuation of multiple valves, a device holder was designed that prevented deformation of the entire device caused by operating the valves. A system was subsequently devised to align each valve and actuator with a precision of better than 20 μm to permit the operation of valves. The developed analytical system was verified by analyzing IL-6 molecules using an enzyme-linked immunosorbent assay. Fluid operations such as sample injection, pL-level aliquot sampling and flow switching were accomplished in this device simply by opening/closing specific valves, and a sample consisting of approximately 1500 IL-6 molecules was successfully detected. This study is expected to significantly improve the usability of nanofluidic analytical devices and lead to the realization of sophisticated analytical techniques such as single-cell proteomics.