Severe infection and sepsis are a common, expensive, and frequently fatal conditions in critically ill patients. The sepsis diagnosis is not trivial, since it is an extremely complex chain of events involving inflammatory and anti-inflammatory processes, cellular reactions, and circulatory disorders. For these reasons, delay in diagnosis and initiation of drug treatments have shown to be crucial for this pathology. Moreover, a multitude of biomarkers has been proposed, many more than for other pathologies. In order to select optimal treatments for the highly heterogeneous group of sepsis patients and to reduce costs, novel multiplexed tools that better characterize the patient and his or her specific immune response are highly desired. In order to achieve the fundament of drastically improved multi-analyte detection and to attain low limits of detection in diagnostics, the area of point-of-care testing (POCT) technology is developing quickly, leading to the production of instruments, the reliability of which is continuously increasing. For this purpose, a selection of two biomarkers—C-reactive protein (CRP) and neopterin (NP)—was studied in this paper and a fluorescence-based integrated optical system, suitable for future POCT applications, was implemented that is capable of performing the simultaneous measurement of the two different biomarkers in replicate. A limit of detection of 10 and 2.1 µg L−1 was achieved for CRP and NP spiked in commercially available human serum, respectively. Moreover, measurements on both biomarkers were also performed on serum samples collected from septic patients.