We have developed a paper and gold nanoparticle (AuNP)-based lab-in-a-syringe (LIS) for immunosensing of biomarkers. This simple diagnostic device features simultaneous sampling and vertical-flow operation, meaning that unlike typical immunosensors, it does not suffer from any delay between sampling and detection. It can handle large-volume, low-concentration samples for sample analysis in diverse applications (e.g. biomedical, environmental, food, etc.). Furthermore, its operating range for sample concentration can by tuned by simply changing the volume of the syringed sample, which enables on-demand limits of detection (LOD). The LIS contains two nitrocellulose pads: the conjugate pad (which captures the analyte) and the detection pad (which signals the presence of the captured analyte) both embedded into reusable plastic cartridges. We demonstrated its efficiency at detecting IgG (LOD: 1.0 ng/mL) and prostate-specific antigen (PSA) (spiked urine samples; LOD: 1.9 ng/mL). In the field the LIS can be used for complete on-site analysis, or to obtain partially analyzed samples (AuNPs with captured analyte) for subsequent detailed testing in specialized laboratories.