In this study, for the first time, we have shown that the single nano-/microparticle trapping ability of optical tweezers combined with the high selectivity of molecularly imprinted polymers (MIPs) provides an indispensable molecular-level instrument for chemical sciences. Trapping a single MIP inside a solution and analyzing its Brownian motion allow for real-time determination of its target molecule [trimipramine (TMP) in our case] content. This method is also utilized to precisely measure TMP concentration in the bulk solution. The detection and optical volumes, respectively, defined as single MIP volume and laser focal volume, were about a few femtoliters. According to our data within a detection volume inside the bulk solution, 0.02-0.25 target molecules could be detectable with a detection limit of 0.005 molecules. Thus, we detected 1/1000th of the subsingle molecule in detection volume by high-resolution densitometry.