Direct laser writing is a lithography technique for fabricating features in sub-micron dimensions. Major advantages of this technique are the elimination of mask, cost-effectiveness and design scalability. Patterns with different dimensions and geometries can be fabricated using this technique, which are greatly influenced by several laser parameters such as the intensity of the laser, speed of the stage (substrate), and focus level of the laser. To achieve high accuracy, the effects of the operative parameters on width are required to be mapped. In this work, the effect of various parameters, such as laser intensity, stage speed, and focus level (Z-axis movement), on the features of the micropatterns has been investigated. The laser intensity was varied from 25 000 W m−2 to 65 000 W m−2, and the stage speed was varied from 20 steps per minute to 200 steps per minute. The focus level was altered from the far limit of the Z-axis and incremented by a value of 0.1 mm. The patterns were created on glass substrates coated with negative dry film photoresist. The geometry of the obtained patterns was analyzed using optical microscopy and scanning electron microscopy. Subsequently, the statistical tools were employed to frame empirical relationships of the parameters with feature size as a response function. The construction of the efficient experimental combinations has been performed using design of experiments and the response values were analyzed using tools such as analysis of means and analysis of variance. Except knowing the significance of all the individual parameters, it was observed that the focus level highly influenced the feature size compared to the intensity of laser and stage speed. The applications of these devices in the fields of droplet based microfluidics and interdigitated electrodes for electrochemical sensing have also been discussed.