The global demand for lithium has soared in recent years due to the wide use of lithium batteries. To meet this demand, we herein report developing novel on‐site sample preparation methods for the extraction of Li+ from relevant materials, including brine water, spodumene rock, and lithium‐ion battery electrodes, and a DNAzyme‐based fluorescent sensor for sensitive and robust detection of Li+ in these samples down to 1.4 mM (10 ppm) using a portable fluorometer. The system can distinguish key threshold lithium levels that indicate economic value across several industries, including 200 ppm Li+ for brine mining, 6% Li2O or SC6 for rock mining, and Li+‐specific aging in LIBs. The methods developed and demonstrated in this work will allow highly selective, on‐site, portable detection of lithium in both environmental samples to identify new lithium resources and in battery electrodes to guide recycling strategies in order to meet the global demand for lithium.