We have developed a colorimetric sensing strategy employing gold nanoparticles and a paper-based analytical platform for the diagnosis of tuberculosis (TB). By utilizing the surface plasmon resonance effect, we were able to monitor changes in the color of a gold nanoparticle colloid based on the effects of single-stranded DNA probe molecules hybridizing with targeted double-stranded TB DNA. The hybridization event changes the surface charge density of the nanoparticles, causing them to aggregate to various degrees, which modifies the color of the solution in a manner that can be readily measured to determine the concentration of the targeted DNA analyte. In order to adapt this TB diagnosis method to resource-limited settings, we extended this label-free oligonucleotide and unmodified gold nanoparticle solution-based technique to a paper-based system that can be measured using a smartphone to obtain rapid parallel colorimetric results with low reagent consumption and without the need for sophisticated analytical equipment. In this study, we investigated various assay conditions, including the denaturing temperature and time, different oligonucleotide probe sequences, as well as the ratio of single stranded probe and double stranded target DNA. After optimizing these variables, we were able to achieve a detection limit of 1.95 × 10^–2 ng/mL for TB DNA. Furthermore, multiple tests could be performed simultaneously with a 60 min turnaround time.