This paper demonstrates a writing and reading methodology, which allows both to create and to detect sub-100-nm carboxyl-terminated patterns on light-transmissive quartz substrates by the same instrumental system. Such a technique, capable of creating carboxyl-terminated nanopatterns, offers several benefits for the miniaturization of biochips, since the carboxyl-terminated nanopatterns allow the easy immobilization of biomolecules by amide bond formation. As a consequence, increasingly miniaturized biochips require suitable analytical methods for the detection of nanopatterns. In our approach, carboxyl-terminated nanopatterns of down to 80 nm width were created using a photolabile silane coupling agent and a UV laser coupled to a near-field scanning optical microscope (NSOM). The same NSOM system was then used in a next step to detect the fabricated carboxyl-terminated nanopatterns after modification with a fluorescent label. Furthermore, as a first step towards biochip applications, the successful immobilization of several biomolecules, such as streptavidin, IgG and DNA on carboxyl-terminated nanopatterns was demonstrated. We have shown that our approach has the potential to lead to a new bioanalytical method, which enables one to write and to read biochips on a sub-100-nm scale by the same system.